Optically-Controlled CNS Dysfunction

ABSTRACT

Provided herein are animals expressing light-responsive opsin proteins in the basal lateral amygdala of the brain and methods for producing the same wherein illumination of the light-responsive opsin proteins causes anxiety in the animal. Also provided herein are methods for alleviating and inducing anxiety in an animal as well as methods for screening for a compound that alleviates anxiety in an animal.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of U.S. provisionalapplication Ser. No. 61/410,748 filed on Nov. 5, 2010, and 61/464,806filed on Mar. 8, 2011, the contents of each of which are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

Anxiety is a sustained state of heightened apprehension in the absenceof immediate threat, which in disease states becomes severelydebilitating. Anxiety disorders represent the most common of thepsychiatric diseases (with 28% lifetime prevalence), and have beenlinked to the etiology of major depression and substance abuse. Whilethe amygdala, a brain region important for emotional processing, haslong been hypothesized to play a role in anxiety, the neural mechanismswhich control and mediate anxiety have yet to be identified. Despite thehigh prevalence and severity of anxiety disorders, the correspondingneural circuit substrates are poorly understood, impeding thedevelopment of safe and effective treatments. Available treatments tendto be inconsistently effective or, in the case of benzodiazepines,addictive and linked to significant side effects including sedation andrespiratory suppression that can cause cognitive impairment and death. Adeeper understanding of anxiety control mechanisms in the mammalianbrain is necessary to develop more efficient treatments that have fewerside-effects. Of particular interest and novelty would be thepossibility of recruiting native pathways for anxiolysis.

SUMMARY OF THE INVENTION

Provided herein is an animal comprising a light-responsive opsinexpressed in glutamatergic pyramidal neurons of the basolateral amygdala(BLA), wherein the selective illumination of the opsin in the BLA-CeLinduces anxiety or alleviates anxiety of the animal.

Provided herein is an animal comprising a light-responsive opsinexpressed in glutamatergic pyramidal neurons of the BLA, wherein theopsin is an opsin which induces hyperpolarization by light, and whereinthe selective illumination of the opsin in the BLA-CeL induces anxietyof the animal. In some embodiments, the opsin is NpHR, BR, AR, or GtR3.In some embodiments, the NpHR comprises the amino acid sequence of SEQID NO:1, 2, or 3. In some embodiments, the animal further comprises asecond light-responsive opsin expressed in glutamatergic pyramidalneurons of the BLA, wherein the second opsin is an opsin that inducesdepolarization by light, and wherein the selective illumination of thesecond opsin in the BLA-CeL reduces anxiety of the animal. In someembodiments, the second opsin is ChR2, VChR1, or DChR. In someembodiments, the second opsin is a C1V1 chimeric protein comprising theamino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments,the second opsin comprises the amino acid sequence of SEQ ID NO:6 or 7.

Provided herein is an animal comprising a light-responsive opsinexpressed in the glutamatergic pyramidal neurons of the BLA, wherein theopsin is an opsin that induces depolarization by light, and wherein theselective illumination of the opsin in the BLA-CeL reduces anxiety ofthe animal. In some embodiments, the opsin is ChR2, VChR1, or DChR. Insome embodiments, the opsin is a C1V1 chimeric protein comprising theamino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments,the opsin comprises the amino acid sequence of SEQ ID NO:6 or 7.

Also provided herein is a vector for delivering a nucleic acid toglutamatergic pyramidal neurons of the BLA in an individual, wherein thevector comprises the nucleic acid encoding a light-responsive opsin andthe nucleic acid is operably linked to a promoter that controls thespecific expression of the opsin in the glutamatergic pyramidal neurons.In some embodiments, the promoter is a CaMKIIα promoter. In someembodiments, the vector is an AAV vector. In some embodiments, the opsinis an opsin that induces depolarization by light, and wherein selectiveillumination of the opsin in the BLA-CeL alleviates anxiety. In someembodiments, the opsin that induces depolarization by light is ChR2,VChR1, or DChR. In some embodiments, the opsin is a C1V1 chimericprotein comprising the amino acid sequence of SEQ ID NO:8, 9, 10, or 11.In some embodiments, the opsin comprises the amino acid sequence of SEQID NO:6 or 7. In some embodiments, the opsin is an opsin that induceshyperpolarization by light, and wherein selective illumination of theopsin in the BLA-CeL and induces anxiety. In some embodiments, the opsinthat induces hyperpolarization by light is NpHR, BR, AR, or GtR3. Insome embodiments, the NpHR comprises the amino acid sequence of SEQ IDNO:1, 2, or 3. In some embodiments, the individual is a mouse or a rat.In some embodiments, the individual is a human.

Also provided here is a method of delivering a nucleic acid toglutamatergic pyramidal neurons of the BLA in an individual, comprisingadministering to the individual an effective amount of a vectorcomprising a nucleic acid encoding a light-responsive opsin and thenucleic acid is operably linked to a promoter that controls the specificexpression of the opsin in the glutamatergic pyramidal neurons. In someembodiments, the promoter is a CaMKIIα promoter. In some embodiments,the vector is an AAV vector. In some embodiments, the opsin is an opsinthat induces depolarization by light, and wherein selective illuminationof the opsin in the BLA-CeL alleviates anxiety. In some embodiments, theopsin that induces depolarization by light is ChR2, VChR1, or DChR. Insome embodiments, the opsin is a C1V1 chimeric protein comprising theamino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments,the opsin comprises the amino acid sequence of SEQ ID NO:6 or 7. In someembodiments, the opsin is an opsin that induces hyperpolarization bylight, and wherein selective illumination of the opsin in the BLA-CeLand induces anxiety. In some embodiments, the opsin that induceshyperpolarization by light is NpHR, BR, AR, or GtR3. In someembodiments, the NpHR comprises the amino acid sequence of SEQ ID NO:1,2, or 3. In some embodiments, the individual is a mouse or a rat. Insome embodiments, the individual is a human.

Also provided herein is a coronal brain tissue slice comprising BLA,CeL, and CeM, wherein a light-responsive opsin is expressed in theglutamatergic pyramidal neurons of the BLA. In some embodiments, theopsin is an opsin that induces depolarization by light. In someembodiments, the opsin that induces depolarization by light is ChR2,VChR1, or DChR. In some embodiments, the opsin is a C1V1 chimericprotein comprising the amino acid sequence of SEQ ID NO:8, 9, 10, or 11.In some embodiments, the opsin comprises the amino acid sequence of SEQID NO:6 or 7. In some embodiments, the opsin is an opsin that induceshyperpolarization by light. In some embodiments, the opsin that induceshyperpolarization by light is NpHR, BR, AR, or GtR3. In someembodiments, the NpHR comprises the amino acid sequence of SEQ ID NO:1,2, or 3. In some embodiments, the tissue is a mouse or a rat tissue.

Also provided herein is a method for screening for a compound thatalleviates anxiety, comprising (a) administering a compound to an animalhaving anxiety induced by selectively illumination of an opsin expressedin the glutamatergic pyramidal neurons of the BLA, wherein the animalcomprises a light-responsive opsin expressed in the glutamatergicpyramidal neurons of the BLA, wherein the opsin is an opsin that induceshyperpolarization by light; and (b) determining the anxiety level of theanimal, wherein a reduction of the anxiety level indicates that thecompound may be effective in treating anxiety. In some embodiments, theopsin is NpHR, BR, AR, or GtR3. In some embodiments, the NpHR comprisesthe amino acid sequence of SEQ ID NO:1, 2, or 3.

Also provided herein is a method for alleviating anxiety in anindividual, comprising: (a) administering to the individual an effectiveamount of a vector comprising a nucleic acid encoding a light-responsiveopsin and the nucleic acid is operably linked to a promoter thatcontrols the specific expression of the opsin in the glutamatergicpyramidal neurons of the BLA, wherein the opsin is expressed in theglutamatergic pyramidal neurons of the BLA, wherein the opsin is anopsin that induces depolarization by light; and (b) selectivelyilluminating the opsin in the glutamatergic pyramidal neurons in theBLA-CeL to alleviate anxiety. In some embodiments, the promoter is aCaMKIIα promoter. In some embodiments, the vector is an AAV vector. Insome embodiments, the opsin is ChR2, VChR1, or DChR. In someembodiments, the opsin is a C1V1 chimeric protein comprising the aminoacid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments, theopsin comprises the amino acid sequence of SEQ ID NO:6 or 7.

Also provided herein is a method for inducing anxiety in an individual,comprising: (a) administering to the individual an effective amount of avector comprising a nucleic acid encoding an opsin and the nucleic acidis operably linked to a promoter that controls the specific expressionof the opsin in the glutamatergic pyramidal neurons of the BLA, whereinthe opsin is expressed in the glutamatergic pyramidal neurons, whereinthe opsin is an opsin that induces hyperpolarization by light; and (b)selectively illuminating the opsin in the glutamatergic pyramidalneurons in the BLA-CeL to induce anxiety. In some embodiments, thepromoter is a CaMKIIα promoter. In some embodiments, the vector is anAAV vector. In some embodiments, the opsin is NpHR, BR, AR, or GtR3. Insome embodiments, the NpHR comprises the amino acid sequence of SEQ IDNO:1, 2, or 3.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Various example embodiments may be more completely understood inconsideration of the following description and the accompanyingdrawings, in which:

FIG. 1 shows a system for providing optogenetic targeting of specificprojections of the brain, consistent with an embodiment of the presentdisclosure; and

FIG. 2 shows a flow diagram for use of an anxiety-based circuit model,consistent with an embodiment of the present disclosure.

FIG. 3 shows that projection-specific excitation of BLA terminals in theCeA induced acute reversible anxiolysis. a) All mice were singly-housedin a high-stress environment for at least 1 week prior to behavioralmanipulations and receive 5-ms light pulses at 20 Hz for all light onconditions. Mice in the ChR2:BLA-CeA group received viral transductionof ChR2 in BLA neurons under the CaMKII promoter and were implanted witha beveled cannula shielding light away from BLA somata to allowselective illumination of BLA terminals in the CeA, while control groupseither received a virus including fluorophore only (EYFP:BLA-CeA group)or a light fiber directed to illuminate BLA somata (ChR2:BLA Somatagroup). (b-c) Mice in the ChR2:BLA-CeA group (n=8) received selectiveillumination of BLA terminals in the CeA during the light on epochduring the elevated plus maze, as seen in this ChR2:BLA-CeArepresentative path (b), which induced a 5-fold increase in open armtime during the light on epoch relative to the light off epochs andEYFP:BLA-CeA (n=9) and ChR2:BLA Somata (n=7) controls (c), as well as asignificant increase in the probability of entering the open arm (seeinset). (d-f) Mice in the ChR2:BLA-CeA group also showed an increase inthe time spent in the center of the open field chamber, as seen in thisrepresentative trace (d), during light on epochs relative to light offepochs and EYFP:BLA-CeA and ChR2:BLA Somata controls (e), but did notshow a significant change in locomotor activity during light on epochs(f). g) Confocal image of a coronal slice showing the CeA and BLAregions from a mouse in the ChR2:BLA-CeA group wherein 125 μm×125 μmsquares indicate regions used for quantification. h) Expanded regionsare arranged in rows by group and in columns by brain region. (i-k)Percent of EYFP-positive and c-fos-positive neurons of allDAPI-identified cells for all groups, by region. Numbers of counted pergroup and region are indicated in legends. None of the regions examinedshowed detectable differences in the proportion of EYFP-positive cellsamong groups. i) Proportion of BLA neurons that were EYFP-positive orc-fos-positive. The ChR2:BLA Somata group had a significantly higherproportion of c-fos-positive BLA neurons (F_(2,9)=10.12, p<0.01)relative to ChR2:BLA-CeA (p<0.01) or EYFP:BLA-CeA (p<0.05) groups. j)The ChR2:BLA-CeA group had a significantly higher proportion ofc-fos-positive cells in the CeL relative to the EYFP:BLA-CeA group(p<0.05), but not the ChR2:BLA Somata group. k) Summary data for CeMneurons show no detectable differences among groups.

FIG. 4 shows projection-specific excitation of BLA terminals in the CeAactivates CeL neurons and elicits feed-forward inhibition of CeMneurons. a) Live two-photon images of representative light-responsiveBLA, CeL and CeM cells all imaged from the same slice, overlaid on abrightfield image. (b-f) Schematics of the recording and illuminationsites for the associated representative current-clamp traces (V_(m)=˜70mV). b) Representative trace from a BLA pyramidal neuron expressingChR2, all BLA neurons expressing ChR2 in the BLA spiked for every 5 mspulse (n=4). c) Representative trace from a CeL neuron in the terminalfield of BLA projection neurons, showing both sub-threshold andsupra-threshold excitatory responses to light-stimulation (n=16). Insetleft, population summary of mean probability of spiking for each pulsein a 40-pulse train at 20 Hz, dotted lines indicate SEM. Inset right,frequency histogram showing individual cell spiking fidelity for 5 mslight pulses delivered at 20 Hz, y-axis is the number of cells per each5% bin. d) Six sweeps from a CeM neuron spiking in response to a currentstep (˜60 pA; indicated in black) and inhibition of spiking upon 20 Hzillumination of BLA terminals in the CeL. Inset, spike frequency wassignificantly reduced during light stimulation of CeL neurons (n=4).(e-f) Upon broad illumination of the CeM, voltage-clamp summaries showthat the latency of EPSCs is significantly shorter than the latency ofIPSCs, while there was a non-significant difference in the amplitude ofEPSCs and IPSCs (n=11; *p=0.04, see insets). The same CeM neurons (n=7)showed either net excitation when receiving illumination of the CeM (e)or net inhibition upon selective illumination of the CeL (f).

FIG. 5 shows light-induced anxiolytic effects were attributable toactivation of BLA-CeL synapses alone. (a-b) 2-photon z-stack images of18 dye-filled BLA neurons were reconstructed, and their projections tothe CeL and CeM are summarized in (a), with their images shown in (b)wherein red indicates projections to CeL, blue indicates projections toCeM and purple indicates projections to both CeL and CeM. c) Schematicof the recording site and the light spot positions, as whole-cellrecordings were performed at each location of the light spot, which wasmoved in 100 um-steps away from the cell soma both over a visualizedaxon and in a direction that was not over an axon. d) Normalizedcurrent-clamp summary of spike fidelity to a 20 Hz train delivered atvarious distances from the soma, showing that at ˜300 um away from thecell soma, illumination of an axon terminal results in low (<5%) spikefidelity. e) Normalized voltage-clamp summary of depolarizing currentseen at the cell soma upon illumination per distance from cell soma.(f-i) Representative current-clamp traces upon illumination with a ˜150um-diameter light spot over various locations within each slicepreparation (n=7). Illumination of the cell soma elicits high-fidelityspiking (f). Illumination of BLA terminals in CeL elicits strong sub-and supra-threshold excitatory responses in the postsynaptic CeL neuron(g), but does not elicit reliable antidromic spiking in the BLA neuronitself (h), and light delivered off axon is shown for comparison as acontrol for light scattering (i). (k-j) A separate group of ChR2:BLA-CeLmice (n=8) were each run twice on the elevated plus maze and the openfield test, one session preceded with intra-CeA infusions of saline(red) and the other session with the glutamate receptor antagonists NBQXand AP5 (purple), counterbalanced for order. k) Glutamate receptorblockade in the CeA attenuated light-induced increases in both timespent in open arms as well as the probability of open arm entry (inset)on the elevated plus maze without impairing performance during light offepochs. j) Local glutamate receptor antagonism significantly attenuatedlight-induced increases in center time on the open field test, insetshows pooled summary.

FIG. 6 shows that selective inhibition of BLA terminals in the CeAinduced an acute and reversible increase in anxiety. a) All mice weregroup-housed in a low-stress environment and received bilateral constant591 nm light during light on epochs. Mice in the eNpHR3.0:BLA-CeA group(n=9) received bilateral viral transduction of eNpHR3.0 in BLA neuronsunder the CaMKII promoter and were implanted with a beveled cannulashielding light away from BLA somata to allow selective illumination ofBLA terminals in the CeA, while control groups either received bilateralvirus transduction of a fluorophore only (EYFP:BLA-CeA bil group; n=8)or a light fiber directed to illuminate BLA somata (eNpHR3.0:BLA Somatagroup; n=6). b) Confocal image of the BLA and CeA of a mouse treatedwith eNpHR3.0. (c-e) In the same animals used in anxiety assays below, asignificantly higher proportion of neurons in the CeM (e) from theeNpHR3.0 group expressed c-fos relative to the EYFP group (*p<0.05). f)Representative path of a mouse in the eNpHR3.0:BLA-CeA group, showing adecrease in open arm exploration on the elevated plus maze during epochsof selective illumination of BLA terminals in the CeA. g) eNpHR3.0 miceshowed a reduction in the time spent in open arms and probability ofopen arm entry (inset) during light stimulation, relative to controls.h) Representative path of a mouse from the eNpHR3.0:BLA-CeA group duringpooled light off and on epochs in the open field test. i) Significantreduction in center time in the open field chamber for theeNpHR3.0:BLA-CeA group during light on, but not light off, epochs ascompared to controls, inset shows pooled data summary. (j−1) Selectiveillumination of eNpHR3.0-expressing BLA terminals is sufficient toreduce spontaneous vesicle release in the presence of carbachol.Representative trace of a CeL neuron (j) from an acute slice preparationin which BLA neurons expressed eNpHR 3.0, shows that when BLA terminals˜300 μm away from the BLA soma are illuminated, there is a reduction inthe amplitude (k) and frequency (1) of sEPSCs seen at the postsynapticCeL neuron. Cumulative distribution frequency of the amplitude (k) andfrequency (l) of sEPSCs recorded at CeL neurons (n=5) upon variouslengths of illumination 5-60 s, insets show respective mean+SEM in theepochs of matched duration before, during and after illumination(**p<0.01; ***p<0.001). (m-p) Selective illumination of BLA terminalsexpressing eNpHR 3.0 suppresses vesicle release evoked by electricalstimulation in the BLA. m) Schematic indicating the locations of thestimulating electrode, the recording electrode and the ˜150 μm diameterlight spot. n) Representative traces of EPSCs in a CeL neuron before(Off₁), during (On) and after (Off₂) selective illumination of BLAterminals expressing eNpHR3.0. Normalized EPSC amplitude summary data(o) and individual cell data (p) from slice preparations containing BLAneurons expressing eNpHR 3.0 (n=7) and non-transduced controls (n=5)show that selectively illuminating BLA terminals in the CeLsignificantly (*p=0.006) reduces the amplitude of electrically-evokedEPSCs in postsynaptic CeL neurons.

FIG. 7 is a diagram showing the histologically verified placements ofmice treated with 473 nm light. Unilateral placements of the virusinjection needle (circle) and the tip of beveled cannula (x) areindicated, counter-balanced for hemisphere. Colors indicate treatmentgroup, see legend. Coronal sections containing the BLA and the CeA areshown here, numbers indicate the anteroposterior coordinates from bregma(Aravanis et al., J Neural Eng, 4:S143-156, 2007).

FIG. 8 shows the beveled cannula and illumination profile design. a)Light cone from bare fiber emitting 473 nm light over cuvette filledwith fluorescein in water. The angle of the light cone is approximately12 degrees. b) Light cone from the same fiber and light ensheathed in abeveled cannula. The beveled cannula blocks light delivery to one side,without detectably altering perpendicular light penetrance. c) Diagramof light delivery via the optical fiber with the beveled cannula overCeA. d) Chart indicating estimated light power density seen at variousdistances from the fiber tip in mouse brain tissue when the light powerdensity seen at the fiber tip was 7 mW (˜99 mW/mm²) Inset, cartoonindicating the configuration. Optical fiber is perpendicular and aimedat the center of the power meter, through a block of mouse brain tissue.e) Table showing light power (mW) as measured by a standard power meterand the estimated light power density (mW/mm²) seen at the tip, at theCeL (˜0.5-0.7 mm depth in brain tissue) and at the CeM (˜1.1 mm depth inbrain tissue).

FIG. 9 demonstrates that the beveled cannula prevented light delivery toBLA and BLA spiking at light powers used for behavioral assays. a)Schematic indicating the configuration of light delivery by opticalfiber to the CeA and recording electrode (red) in the BLA. b)Scatterplot summary of recordings in the BLA with various light powersdelivered to the CeA with and without the beveled cannula (n=4 sites).For each site, repeated alternations of recordings were made with andwithout the beveled cannula. The x-axis shows both the light powerdensity at the fiber tip (black) and the estimated light power densityat the CeL (grey). The blue vertical or shaded region indicates therange of light power densities used for behavioral assays (˜7 mW; ˜99mW/mm² at the tip of the fiber). Reliable responses from BLA neuronswere not observed in this light power density range. c) Representativetraces of BLA recordings with 20 Hz 5 ms pulse light stimulation at 7 mW(˜99 mW/mm² at fiber tip; ˜5.9 mW/mm² at CeL) at the same recording sitein the CeA. d) Population spike waveforms in response to single pulsesof light reveal substantial light restriction even at high 12 mV power(˜170 mW/mm² at the tip of the fiber; ˜10.1 mW/mm² at CeL).

FIG. 10 demonstrates that viral transduction excluded intercalated cellclusters. a) Schematic of the intercalated cells displayed in subsequentconfocal images. (b-d) Representative images of intercalated cells frommice that received EYFP b), eNpHR 3.0 c) and ChR2 d) injections into theBLA that were used for behavioral manipulations. Viral expression wasnot observed in intercalated cell clusters. (e-f) There were very low(<2%) levels of YFP expression in intercalated cell clusters for all 6groups used in behavioral assays. There were no statisticallysignificant differences among groups in c-fos expression.

FIG. 11 shows that unilateral intra-CeA administration of glutamateantagonists did not alter locomotor activity. Administration of NBQX andAP5 prior to the open field test did not impair locomotor activity (asmeasured by mean velocity) relative to saline infusion (F_(1,77)=2.34,p=0.1239).

FIG. 12 demonstrates that bath application of glutamate antagonistsblocked optically-evoked synaptic transmission. 4-6 weeks followingintra-BLA infusions of AAV5-CamKII-ChR2-EYFP into the BLA of wild-typemice, we examined the ability of the glutamate receptor antagonists NBQXand AP5 to block glutamatergic transmission. a) Representativecurrent-clamp (top) and voltage-clamp (bottom) traces of arepresentative CeL neuron upon a 20 Hz train of 473 nm lightillumination of BLA terminals expressing ChR2. b) The same cell'sresponses following bath application of NBQX and AP5 show abolishedspiking and EPSCs. c) Population summary (n=5) of the depolarizingcurrent seen before and after bath application of NBQX and AP5,normalized to the pre-drug response.

FIG. 13 is a diagram depicting the histologically verified placements ofmice treated with 594 nm light. Bilateral placements of virus injectionneedle (circle) and tip of beveled cannula (x) are indicated. Colorsindicate treatment group, see legend. Coronal sections containing BLAand CeA are shown; numbers indicate AP coordinates from bregma (Aravaniset el., J Neural Eng, 4:S143-156, 2007).

FIG. 14 shows that light stimulation parameters used in the eNpHR 3.0terminal inhibition experiments does not block spiking at the cell soma.(a-c) Schematics of the light spot location and recording sitesalongside corresponding representative traces upon a current steplasting the duration of the spike train, paired with yellow lightillumination at each location during the middle epoch (indicated byyellow horizontal bar). a) Representative current-clamp trace from a BLAneuron expressing eNpHR 3.0 upon direct illumination shows potentinhibition of spiking during illumination of cell soma. b)Representative current-clamp trace from a BLA neuron expressing eNpHR3.0 when a ˜125 μm diameter light spot is presented ˜300 μm away fromthe cell soma without illuminating an axon. c) Representativecurrent-clamp trace from a BLA neuron expressing eNpHR 3.0 when a ˜125μm diameter light spot is presented ˜300 μm away from the cell soma whenilluminating an axon. d) While direct illumination of the cell somainduced complete inhibition of spiking that was significant from allother conditions (F_(3,9)=81.50, p<0.0001; n=3 or more per condition),there was no significant difference among the distal illumination ˜300μm away from the soma of BLA neurons expressing eNpHR 3.0 conditions andthe no light condition (F_(2,7)=0.79, p=0.49), indicating that distalillumination did not significantly inhibit spiking at the cell soma. e)Schematic indicating light spot locations relative to recording site,regarding the population summary shown to the right. Population summaryshows the normalized hyperpolarizing current recorded from the cell somaper distance of light spot from cell soma, both on and off axoncollaterals (n=5).

FIG. 15 demonstrates that selective illumination of BLA terminalsinduced vesicle release onto CeL neurons without reliably elicitingantidromic action potentials. Schematics and descriptions refer to thetraces below, and trace color indicates cell type. Light illuminationpatterns are identical for both series of traces. Left column, CeLtraces for three overlaid sweeps of a 40-pulse light train per cell(n=8). Here, both time-locked EPSCs indicate vesicle release from thepresynaptic ChR2-expressing BLA terminal, and for all postsynaptic CeLcells, there were excitatory responses to 100% of light pulses. Rightcolumn, BLA traces for three overlaid 40-pulse sweeps per cell (n=9),with the mean number of light pulses delivered at the axon terminalresulting in a supra-threshold antidromic action potential (5.4%±2%,mean±SEM).

FIG. 16 is a graph demonstrating that light stimulation did not alterlocomotor activity in eNpHR 3.0 and control groups. There were nodetectable differences in locomotor activity among groups nor lightepochs (F_(1,20)=0.023, p=0.3892; F_(1,100)=3.08, p=0.086).

DETAILED DESCRIPTION

The present disclosure relates to control over nervous system disorders,such as disorders associated with anxiety and anxiety symptoms, asdescribed herein. While the present disclosure is not necessarilylimited in these contexts, various aspects of the invention may beappreciated through a discussion of examples using these and othercontexts.

Various embodiments of the present disclosure relate to an optogeneticsystem or method that correlates temporal control over a neural circuitwith measurable metrics. For instance, various metrics or symptoms mightbe associated with a neurological disorder exhibiting various symptomsof anxiety. The optogenetic system targets a neural circuit within apatient for selective control thereof. The optogenetic system involvesmonitoring the patient for the metrics or symptoms associated with theneurological disorder. In this manner, the optogenetic system canprovide detailed information about the neural circuit, its functionand/or the neurological disorder.

Consistent with the embodiments discussed herein, particular embodimentsrelate to studying and probing disorders. Other embodiments relate tothe identification and/or study of phenotypes and endophenotypes. Stillother embodiments relate to the identification of treatment targets.

Aspects of the present disclosure are directed to using anartificially-induced anxiety state for the study of anxiety in otherwisehealthy animals. This can be particularly useful for monitoring symptomsand aspects that are poorly understood and otherwise difficult toaccurately model in living animals. For instance, it can be difficult totest and/or study anxiety states due to the lack of available animalsexhibiting the anxiety state. Moreover, certain embodiments allow forreversible anxiety states, which can be particularly useful inestablishing baseline/control points for testing and/or for testing theeffects of a treatment on the same animal when exhibiting the anxietystate and when not exhibiting the anxiety state. The reversible anxietystates of certain embodiments can also allow for a reset to baselinebetween testing the effects of different treatments on the same animal.

Certain aspects of the present disclosure are directed to a methodrelated to control over anxiety and/or anxiety symptoms in a livinganimal. In certain more specific embodiments, the monitoring of thesymptoms also includes assessing the efficacy of the stimulus inmitigating the symptoms of anxiety. Various other methods andapplications exist, some of which are discussed in more detail herein.

Light-responsive opsins that may be used in the present inventionincludes opsins that induce hyperpolarization in neurons by light andopsins that induce depolarization in neurons by light. Examples ofopsins are shown in Tables 1 and 2 below.

Table 1 shows identified opsins for inhibition of cellular activityacross the visible spectrum:

TABLE 1 Fast optogenetics: inhibition across the visible spectrumBiological Wavelength Opsin Type Origin Sensitivity Defined action NpHRNatronomonas 589 nm max Inhibition pharaonis (hyperpolarization) BRHalobacterium 570 nm max Inhibition helobium (hyperpolarization) ARAcetabulaira 518 nm max Inhibition acetabulum (hyperpolarization) GtR3Guillardia 472 nm max Inhibition theta (hyperpolarization) MacLeptosphaeria 470-500 nm max Inhibition maculans (hyperpolarization)NpHr3.0 Natronomonas 680 nm utility Inhibition pharaonis 589 nm max(hyperpolarization) NpHR3.1 Natronomonas 680 nm utility Inhibitionpharaonis 589 nm max (hyperpolarization)Table 2 shows identified opsins for excitation and modulation across thevisible spectrum:

TABLE 2 Fast optogenetics: excitation and modulation across the visiblespectrum Wavelength Opsin Type Biological Origin Sensitivity Definedaction VChR1 Volvox carteri 589 nm utility Excitation 535nm max(depolarization) DChR Dunaliella sauna 500 nm max Excitation(depolarization) ChR2 Chlamydomonas 470 nm max Excitation reinhardtii380-405 nm utility (depolarization) ChETA Chlamydomonas 470 nm maxExcitation reinhardtii 380-405 nm utility (depolarization) SFOChlamydomonas 470 nm max Excitation reinhardtii 530 nm max(depolarization) Inactivation SSFO Chlamydomonas 445 nm max Step-likeactivation reinhardtii 590 nm; (depolarization) 390-400 nm InactivationC1V1 Volvox carteri and 542 nm max Excitation Chlamydomonas(depolarization) reinhardtii C1V1 E122 Volvox carteri and 546 nm maxExcitation Chlamydomonas (depolarization) reinhardtii C1V1 E162 Volvoxcarteri and 542 nm max Excitation Chlamydomonas (depolarization)reinhardtii C1V1 E122/ Volvox carteri and 546 nm max Excitation E162Chlamydomonas (depolarization) reinhardtii

Table 2 (Continued):

As used herein, a light-responsive opsin (such as NpHR, BR, AR, GtR3,Mac, ChR2, VChR1, DChR, and ChETA) includes naturally occurring proteinand functional variants, fragments, fusion proteins comprising thefragments or the full length protein. For example, the signal peptidemay be deleted. A variant may have an amino acid sequence at least aboutany of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the naturally occurring protein sequence. A functionalvariant may have the same or similar hyperpolarization function ordepolarization function as the naturally occurring protein.

In some embodiments, the NpHR is eNpHR3.0 or eNpHR3.1 (Seewww.stanford.edu/group/dlab/optogenetics/sequence_info.html). In someembodiments, the light-responsive opsin is a C1V1 chimeric protein or aC1V1-E162 (SEQ ID NO:10), C1V1-E122 (SEQ ID NO:9), or C1V1-E122/E162(SEQ ID NO:11) mutant chimeric protein (See, Yizhar et al, Nature, 2011,477(7363):171-78 andwww.stanford.edu/group/dlab/optogenetics/sequence_info.html). In someembodiments, the light-responsive opsin is a SFO (SEQ ID NO: 6) or SSFO(SEQ ID NO: 7) (See, Yizhar et al, Nature, 2011, 477(7363):171-78;Berndt et al., Nat. Neurosci., 12(2):229-34 andwww.stanford.edu/group/dlab/optogenetics/sequence_info.html).

In some embodiments, the light-activated protein is a NpHR opsincomprising an amino acid sequence at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identical to the sequence shownin SEQ. ID NO:1. In some embodiments, the NpHR opsin further comprisesan endoplasmic reticulum (ER) export signal and/or a membranetrafficking signal. For example, the NpHR opsin comprises an amino acidsequence at least 95% identical to the sequence shown in SEQ ID NO:1 andan endoplasmic reticulum (ER) export signal. In some embodiments, theamino acid sequence at least 95% identical to the sequence shown in SEQID NO:1 is linked to the ER export signal through a linker. In someembodiments, the ER export signal comprises the amino acid sequenceFXYENE, where X can be any amino acid. In another embodiment, the ERexport signal comprises the amino acid sequence VXXSL, where X can beany amino acid. In some embodiments, the ER export signal comprises theamino acid sequence FCYENEV. In some embodiments, the NpHR opsincomprises an amino acid sequence at least 95% identical to the sequenceshown in SEQ ID NO:1, an ER export signal, and a membrane traffickingsignal. In other embodiments, the NpHR opsin comprises, from theN-terminus to the C-terminus, the amino acid sequence at least 95%identical to the sequence shown in SEQ ID NO:1, the ER export signal,and the membrane trafficking signal. In other embodiments, the NpHRopsin comprises, from the N-terminus to the C-terminus, the amino acidsequence at least 95% identical to the sequence shown in SEQ ID NO:1,the membrane trafficking signal, and the ER export signal. In someembodiments, the membrane trafficking signal is derived from the aminoacid sequence of the human inward rectifier potassium channel K_(ir)2.1.In some embodiments, the membrane trafficking signal comprises the aminoacid sequence K S R I T S E G E Y I P L D Q I D I N V. In someembodiments, the membrane trafficking signal is linked to the amino acidsequence at least 95% identical to the sequence shown in SEQ ID NO:1 bya linker. In some embodiments, the membrane trafficking signal is linkedto the ER export signal through a linker. The linker may comprise any of5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,400, or 500 amino acids in length. The linker may further comprise afluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments, thelight-activated opsin further comprises an N-terminal signal peptide. Insome embodiments, the light-activated opsin comprises the amino acidsequence of SEQ ID NO:2. In some embodiments, the light-activatedprotein comprises the amino acid sequence of SEQ ID NO:3.

In some embodiments, the light-activated opsin is a chimeric proteinderived from VChR1 from Volvox carteri and ChR1 from Chlamydomonasreinhardti. In some embodiments, the chimeric protein comprises theamino acid sequence of VChR1 having at least the first and secondtransmembrane helices replaced by the corresponding first and secondtransmembrane helices of ChR1. In other embodiments, the chimericprotein comprises the amino acid sequence of VChR1 having the first andsecond transmembrane helices replaced by the corresponding first andsecond transmembrane helices of ChR1 and further comprises at least aportion of the intracellular loop domain located between the second andthird transmembrane helices replaced by the corresponding portion fromChR1. In some embodiments, the entire intracellular loop domain betweenthe second and third transmembrane helices of the chimericlight-activated protein can be replaced with the correspondingintracellular loop domain from ChR1. In some embodiments, thelight-activated chimeric protein comprises an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:8 without the signalpeptide sequence. In some embodiments, the light-activated chimericprotein comprises an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown inSEQ ID NO:8. C1V1 chimeric light-activated opsins that may have specificamino acid substitutions at key positions throughout the retinal bindingpocket of the VChR1 portion of the chimeric polypeptide. In someembodiments, the C1V1 protein has a mutation at amino acid residue E122of SEQ ID NO:8. In some embodiments, the C1V1 protein has a mutation atamino acid residue E162 of SEQ ID NO:8. In other embodiments, the C1V1protein has a mutation at both amino acid residues E162 and E122 of SEQID NO:8. In some embodiments, each of the disclosed mutant C1V1 chimericproteins can have specific properties and characteristics for use indepolarizing the membrane of an animal cell in response to light.

As used herein, a vector comprises a nucleic acid encoding alight-responsive opsin described herein and the nucleic acid is operablylinked to a promoter that controls the specific expression of the opsinin the glutamatergic pyramidal neurons. Any vectors that are useful fordelivering a nucleic acid to glutamatergic pyramidal neurons may beused. Vectors include viral vectors, such as AAV vectors, retroviralvectors, adenoviral vectors, HSV vectors, and lentiviral vectors.Examples of AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16. ACaMKIIα promoter and any other promoters that can control the expressionof the opsin in the glutamatergic pyramidal neurons may be used.

An “individual” is a mammal, such as a human. Mammals also include, butare not limited to, farm animals, sport animals, pets (such as cats,dogs, horses), primates, mice and rats. An “animal” is a non-humanmammal.

As used herein, “treatment” or “treating” or “alleviation” is anapproach for obtaining beneficial or desired results including andpreferably clinical results. For purposes of this invention, beneficialor desired clinical results include, but are not limited to, one or moreof the following: showing observable and/or measurable reduction in oneor more signs of the disease (such as anxiety), decreasing symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, and/or delaying the progression of thedisease.

As used herein, an “effective dosage” or “effective amount” of a drug,compound, or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results. For therapeutic use, beneficial ordesired results include clinical results such as decreasing one or moresymptoms resulting from the disease, increasing the quality of life ofthose suffering from the disease, decreasing the dose of othermedications required to treat the disease, enhancing effect of anothermedication such as via targeting, and/or delaying the progression of thedisease. As is understood in the clinical context, an effective dosageof a drug, compound, or pharmaceutical composition may or may not beachieved in conjunction with another drug, compound, pharmaceuticalcomposition, or another treatment. Thus, an “effective dosage” may beconsidered in the context of administering one or more therapeuticagents or treatments, and a single agent may be considered to be givenin an effective amount if, in conjunction with one or more other agentsor treatments, a desirable result may be or is achieved.

The above overview is not intended to describe each illustratedembodiment or every implementation of the present disclosure.

DETAILED DESCRIPTION AND EXAMPLE EXPERIMENTAL EMBODIMENTS

The present disclosure is believed to be useful for controlling anxietystates and/or symptoms of anxiety. Specific applications of the presentinvention relate to optogenetic systems or methods that correlatetemporal, spatio and/or cell-type control over a neural circuitassociated with anxiety states and/or symptoms thereof. As many aspectsof the example embodiments disclosed herein relate to and significantlybuild on previous developments in this field, the following discussionsummarizes such previous developments to provide a solid understandingof the foundation and underlying teachings from which implementationdetails and modifications might be drawn, including those found in theExamples. It is in this context that the following discussion isprovided and with the teachings in the references incorporated herein byreference. While the present invention is not necessarily limited tosuch applications, various aspects of the invention may be appreciatedthrough a discussion of various examples using this context.

Anxiety refers to a sustained state of heightened apprehension in theabsence of an immediate threat, which in disease states becomes severelydebilitating. Embodiments of the present disclosure are directed towardthe use of one or more of cell type-specific optogenetic tools withtwo-photon microscopy, electrophysiology, and anxiety assays to studyand develop treatments relating to neural circuits underlyinganxiety-related behaviors.

Aspects of the present disclosure are related to the optogenetictargeting of specific projections of the brain, rather than cell types,in the study of neural circuit function relevant to psychiatric disease.

Consistent with particular embodiments of the present disclosure,temporally-precise optogenetic stimulation of basolateral amygdala (BLA)terminals in the central nucleus of the amygdala (CeA) are used toproduce a reversible anxiolytic effect. The optogenetic stimulation canbe implemented by viral transduction of BLA with a light-responsiveopsin, such as ChR2, followed by restricted illumination in downstreamCeA.

Consistent with other embodiments of the present disclosure, optogeneticinhibition of the basolateral amygdala (BLA) terminals in the centralnucleus of the amygdala (CeA) are used to increase anxiety-relatedbehaviors. The optogenetic stimulation can be implemented by viraltransduction of BLA with a light-responsive opsin, such as eNpHR3.0,followed by restricted illumination in downstream CeA.

Embodiments of the present disclosure are directed towards the specifictargeting of neural cell populations, as anxiety-based effects were notobserved with direct optogenetic control of BLA somata. For instance,targeting of specific BLA-CeA projections as circuit elements have beenexperimentally shown to be sufficient for endogenous anxiety control inthe mammalian brain.

Consistent with embodiments of the present disclosure, the targeting ofthe specific BLA-CeA projections as circuit elements is based upon anumber of factors discussed in more detail hereafter. The amygdala iscomposed of functionally and morphologically heterogeneous subnucleiwith complex interconnectivity. A primary subdivision of the amygdala isthe basolateral amygdala complex (BLA), which encompasses the lateral(LA), basolateral (BL) and basomedial (BM) amygdala nuclei (˜90% of BLAneurons are glutamatergic). In contrast, the central nucleus of theamygdala (CeA), which is composed of the centrolateral (CeL) andcentromedial (CeM) nuclei, is predominantly (˜95%) comprised ofGABAergic medium spiny neurons. The BLA is ensheathed in dense clustersof GABAergic intercalated cells (ITCs), which are functionally distinctfrom both local interneurons and the medium spiny neurons of the CeA.The primary output nucleus of the amygdala is the CeM, which, whenchemically or electrically excited, is believed to mediate autonomic andbehavioral responses that are associated with fear and anxiety viaprojections to the brainstem. While the CeM is not directly controlledby the primary amygdala site of converging environmental and cognitiveinformation (LA), LA and BLA neurons excite GABAergic CeL neurons, whichcan provide feed-forward inhibition onto CeM “output” neurons and reduceamygdala output. The BLA-CeL-CeM is a less-characterized pathwaysuggested to be involved not in fear extinction but in conditionedinhibition. The suppression of fear expression, possibly due to explicitunpairing of the tone and shock, suggested to be related to thepotentiation of BLA-CeL synapses.

BLA cells have promiscuous projections throughout the brain, includingto the bed nucleus of the stria terminalis (BNST), nucleus accumbens,hippocampus and cortex. Aspects of the present disclosure relate tomethods for selective control of BLA terminals in the CeL, withoutlittle or no direct affect/control of other BLA projections.Preferential targeting of BLA-CeL synapses can be facilitated byrestricting opsin gene expression to BLA glutamatergic projectionneurons and by restricting light delivery to the CeA.

For instance, control of BLA glutamatergic projection neurons can beachieved with an adeno-associated virus (AAV5) vector carryinglight-activated optogenetic control genes under the control of a CaMKIIαpromoter. Within the BLA, CaMKIIα is only expressed in glutamatergicpyramidal neurons, not in local interneurons or intercalated cells.

FIG. 1 shows a system for providing optogenetic targeting of specificprojections of the brain, consistent with an embodiment of the presentdisclosure. For instance, a beveled guide cannula can be used to directlight, e.g., prevent light delivery to the BLA and allow selectiveillumination of the CeA. This preferential delivery of light to the CeAprojection can be accomplished using stereotaxic guidance along withimplantation over the CeL. Geometric and functional properties of theresulting light distribution can be quantified both in vitro and invivo, e.g., using in vivo electrophysiological recordings to determinelight power parameters for selective control of BLA terminals but notBLA cell bodies. Experimental results, such as those described in theExamples, support that such selective excitation or inhibition result insignificant, immediate and reversible anxiety-based effects.

Embodiments of the present disclosure are directed toward the aboverealization being applied to various ones of the anatomical, functional,structural, and circuit targets identified herein. For instance, thecircuit targets can be studied to develop treatments for the psychiatricdisease of anxiety. These treatments can include, as non-limitingexamples, pharmacological, electrical, magnetic, surgical andoptogenetic, or other treatment means.

FIG. 2 shows a flow diagram for use of an anxiety-based circuit model,consistent with an embodiment of the present disclosure. An optogeneticdelivery device, such as a. viral delivery device, is generated 202.This delivery device can be configured to introduce optically responsiveopsins to the target cells and may include targeted promoters forspecific cell types. The delivery device can then be stereotaxically (orotherwise) injected 204 into the BLA. A light delivery device can thenbe surgical implanted 206. This light delivery device can be configuredto provide targeted illumination (e.g., using a directional opticalelement). The target area is then illuminated 208. The target area canbe, for example, the BLA-CeA. The effects thereof can then be monitoredand/or assessed 210. This can also be used in connection with treatmentsor drug screening.

Various embodiments of the present disclosure relate to the use of theidentified model for screening new treatments for anxiety. For instance,anxiety can be artificially induced or repressed using the methodsdiscussed herein, while pharmacological, electrical, magnetic, surgical,or optogenetic treatments are then applied and assessed. In otherembodiments of the present disclosure, the model can be used to developan in vitro approximation or simulation of the identified circuit, whichcan then be used in the screening of devices, reagents, tools,technologies, methods and approaches and for studying and probinganxiety and related disorders. This study can be directed towards, butnot necessarily limited to, identifying phenotypes, endophenotypes, andtreatment targets.

Embodiments of the present disclosure are directed toward modeling theBLA-CeL pathway as an endogenous neural substrate for bidirectionallymodulating the unconditioned expression of anxiety. Certain embodimentsare directed toward other downstream circuits, such as CeA projectionsto the BNST, for their role in the expression of anxiety oranxiety-related behaviors. For instance, it is believed thatcorticotropin releasing hormone (CRH) networks in the BNST may becritically involved in modulating anxiety-related behaviors, as the CeLis a primary source of CRH for the BNST. Other neurotransmitters andneuromodulators may modulate or gate effects on distributed neuralcircuits, including serotonin, dopamine, acetylcholine, glycine, GABAand CRH. Still other embodiments are directed toward control of theneural circuitry converging to and diverging from this pathway, asparallel or downstream circuits of the BLA-CeL synapse are believed tocontribute to the modulation or expression of anxiety phenotypes.Moreover, upstream of the amygdala, this microcircuit is well-positionedto be recruited by top-down cortical control from regions important forprocessing fear and anxiety, including the prelimbic, infralimbic andinsular cortices that provide robust innervation to the BLA and CeL.

Experimental results based upon the BLA anatomy suggest that thepopulations of BLA neurons projecting to CeL and CeM neurons are largelynon-overlapping. In natural states, the CeL-projecting BLA neurons mayexcite CeM-projecting BLA neurons in a microcircuit homeostaticmechanism, which can then be used to study underlying anxiety disorderswhen there are synaptic changes that skew the balance of the circuit toallow uninhibited CeM activation.

The embodiments and specific applications discussed herein (includingthe Examples) may be implemented in connection with one or more of theabove-described aspects, embodiments and implementations, as well aswith those shown in the figures and described below. Reference may bemade to the following Example, which is fully incorporated herein byreference. For further details on light-responsive molecules and/oropsins, including methodology, devices and substances, reference mayalso be made to the following background publications: U.S. PatentPublication No. 2010/0190229, entitled “System for Optical Stimulationof Target Cells” to Zhang et al.; U.S. Patent Publication No.2010/0145418, also entitled “System for Optical Stimulation of TargetCells” to Zhang et al.; U.S. Patent Publication No. 2007/0261127,entitled “System for Optical Stimulation of Target Cells” to Boyden etal.; and PCT WO 2011/116238, Entitled “Light Sensitive Ion PassingMolecules”. These applications form part of the patent document and arefully incorporated herein by reference. Consistent with thesepublications, numerous opsins can be used in mammalian cells in vivo andin vitro to provide optical stimulation and control of target cells. Forexample, when ChR2 is introduced into an electrically-excitable cell,such as a neuron, light activation of the ChR2 channelrhodopsin canresult in excitation and/or firing of the cell. In instances when NpHRis introduced into an electrically-excitable cell, such as a neuron,light activation of the NpHR opsin can result in inhibition of firing ofthe cell. These and other aspects of the disclosures of theabove-referenced patent applications may be useful in implementingvarious aspects of the present disclosure.

While the present disclosure is amenable to various modifications andalternative forms, specifics thereof have been shown by way of examplein the drawings and will be described in further detail. It should beunderstood that the intention is not to limit the disclosure to theparticular embodiments and/or applications described. On the contrary,the intention is to cover all modifications, equivalents, andalternatives falling within the spirit and scope of the presentdisclosure.

Examples Introduction

Anxiety is a sustained state of heightened apprehension in the absenceof immediate threat, which in disease states becomes severelydebilitating¹. Anxiety disorders represent the most common of thepsychiatric diseases (with 28% lifetime prevalence)², and have beenlinked to the etiology of major depression and substance abuse³⁻⁵. Whilethe amygdala, a brain region important for emotional processing⁹⁻¹⁷, haslong been hypothesized to play a role in anxiety¹⁸⁻²³, the neuralmechanisms which control and mediate anxiety have yet to be identified.Here, we combine cell type-specific optogenetic tools with two-photonmicroscopy, electrophysiology, and anxiety assays in freely-moving miceto identify neural circuits underlying anxiety-related behaviors.Capitalizing on the unique capability of optogenetics²⁴⁻²⁶ to controlnot only cell types, but also specific connections between cells, weobserved that temporally-precise optogenetic stimulation of basolateralamygdala (BLA) terminals in the central nucleus of the amygdala (CeA),resolved by viral transduction of BLA with ChR2 followed by restrictedillumination in downstream CeA, exerted a profound, immediate, andreversible anxiolytic effect. Conversely, selective optogeneticinhibition of the same defined projection with eNpHR3.0²⁵ potently,swiftly, and reversibly increased anxiety-related behaviors.Importantly, these effects were not observed with direct optogeneticcontrol of BLA somata themselves. Together, these results implicatespecific BLA-CeA projections as circuit elements both necessary andsufficient for endogenous anxiety control in the mammalian brain, anddemonstrate the importance of optogenetically targeting specificprojections, rather than cell types, in the study of neural circuitfunction relevant to psychiatric disease.

Despite the high prevalence and severity¹ of anxiety disorders, thecorresponding neural circuit substrates are poorly understood, impedingthe development of safe and effective treatments. Available treatmentstend to be inconsistently effective or, in the case of benzodiazepines,addictive and linked to significant side effects including sedation andrespiratory suppression that can cause cognitive impairment anddeath^(27,28). A deeper understanding of anxiety control mechanisms inthe mammalian brain^(29,30) is necessary to develop more efficienttreatments that have fewer side-effects. Of particular interest andnovelty would be the possibility of recruiting native pathways foranxiolysis.

The amygdala is critically involved in processing associations betweenneutral stimuli and positive or negative outcomes, and has also beenimplicated in processing unconditioned emotional states. While theamygdala microcircuit has been functionally dissected in the context offear conditioning, amygdalar involvement has been implicated in amultitude of other functions and emotional states, includingunconditioned anxiety. The amygdala is composed of functionally andmorphologically heterogeneous subnuclei with complex interconnectivity.A primary subdivision of the amygdala is the basolateral amygdalacomplex (BLA), which encompasses the lateral (LA), basolateral (BL) andbasomedial (BM) amygdala nuclei (˜90% of BLA neurons areglutamatergic)^(33,34). In contrast, the central nucleus of the amygdala(CeA), which is composed of the centrolateral (CeL) and centromedial(CeM) nuclei, is predominantly (˜95%) comprised of GABAergic mediumspiny neurons³⁵. The BLA is ensheathed in dense clusters of GABAergicintercalated cells (ITCs), which are functionally distinct from bothlocal interneurons and the medium spiny neurons of the CeA^(36, 37). Theprimary output nucleus of the amygdala is the CeM,^(32, 35, 38-40) whichwhen chemically or electrically excited mediates autonomic andbehavioral responses associated with fear and anxiety via projections tothe brainstem^(6, 12, 32, 35). While the CeM is not directly controlledby the primary amygdala site of converging environmental and cognitiveinformation (LA)^(12, 38, 41), LA and BLA neurons excite GABAergic CeLneurons⁴² which can provide feed-forward inhibition onto CeM^(40, 46)“output” neurons and reduce amygdala output. The BLA-CeL-CeM is aless-characterized pathway suggested to be involved not in fearextinction but in conditioned inhibition, the suppression of fearexpression due to explicit unpairing of the tone and shock, due to thepotentiation of BLA-CeL synapses⁴⁷. Although fear is characterized to bea phasic state triggered by an external cue, while anxiety is asustained state that may occur in the absence of an external trigger, wewondered if circuits modulating conditioned inhibition of fear mightalso be involved in modulating unconditioned inhibition of anxiety.

Materials and Methods

Subjects: Male C57BL/6 mice, aged 4-6 weeks at the start of experimentalprocedures, were maintained with a reverse 12-hr light/dark cycle andgiven food and water ad libitum. Animals shown in FIGS. 3, 4 and 5 (micein the ChR2 Terminals, EYFP Terminals and ChR2 Cell Bodies groups) wereall single-housed in a typical high-traffic mouse facility to increasebaseline anxiety levels. Each mouse belonged to a single treatmentgroup. Animals shown in FIG. 6 (Bilateral EYFP and eNpHR 3.0 groups)were group-housed in a special low-traffic facility to decrease baselineanxiety levels. Animal husbandry and all aspects of experimentalmanipulation of our animals were in accordance with the guidelines fromthe National Institute of Health and have been approved by members ofthe Stanford Institutional Animal Care and Use Committee.

Optical Intensity Measurements: Light transmission measurements wereconducted with blocks of brain tissue from acutely sacrificed mice. Thetissue was then placed over the photodetector of a power meter(ThorLabs, Newton, N.J.) to measure the light power of the laserpenetrated the tissue. The tip of a 300 um diameter optical fiber wascoupled to a 473 nm blue laser (OEM Laser Systems, East Lansing, Mich.).To characterize the light transmission to the opposite side of thebevel, the photodetector of the power meter was placed parallel to thebeveled cannula. For visualization of the light cone, we usedFluorescein isothiocyanate-dextran (FD150s; Sigma, Saint Louis, Mo.) atapproximately 5 mg/ml placed in a cuvette with the optical fibers eitherwith or without beveled cannula shielding aimed perpendicularly over thefluorescein solution. Power density at specific depths were calculatedconsidering both fractional decrease in intensity due to the conicaloutput of light from the optical fiber and the loss of light due toscattering in tissue (Aravanis et al., J Neural Eng, 4:S143-156, 2007)(Gradinaru et al., J Neurosci, 27:14231-14238, 2007). The half-angle ofdivergence θ_(div) for a multimode optical fiber, which determines theangular spread of the output light, is

$\theta_{div} = {\sin^{- 1}\left( \frac{{NA}_{fib}}{n_{tis}} \right)}$

where n_(tis) is the index of refraction of gray matter (1.36, Vo-Dinh T2003, Biomedical Photonics Handbook (Boca Raton, Fla.: CRC Press)) andNA_(fib)(0.37) is the numerical aperture of the optical fiber. Thefractional change in intensity due to the conical spread of the lightwith distance (z) from the fiber end was calculated using trigonometry

${\frac{I(z)}{I\left( {z = 0} \right)} = \frac{\rho^{2}}{\left( {z + \rho} \right)^{2}}},{{{where}\mspace{14mu} \rho} = {r\sqrt{\left( \frac{n}{NA} \right)^{2} - 1}}}$

and r is the radius of the optical fiber (100 μm).

The fractional transmission of light after loss due to scattering wasmodeled as a hyperbolic function using empirical measurements and theKubelka-Munk model^(1, 2), and the combined product of the power densityat the tip of the fiber and the fractional changes due to the conicalspread and light scattering, produces the value of the power density ata specific depth below the fiber.

Virus construction and packaging: The recombinant AAV vectors wereserotyped with AAV₅ coat proteins and packaged by the viral vector coreat the University of North Carolina. Viral titers were 2×10 e¹²particles/mL, 3×10 e¹² particles/mL, 4×10 e¹² particles/mL respectivelyfor AAV-CaMKIIα-hChR2(H134R)-EYFP, AAV-CaMKIIα-EYFP, andAAV-CaMKIIα-eNpHR 3.0-EYFP. The pAAV-CaMKIIα-eNpHR3.0-EYFP plasmid wasconstructed by cloning CaMKIIα-eNpHR3.0-EYFP into an AAV backbone usingMluI and EcoRI restriction sites. Similarly, The pAAV-CaMKIIα-EYFPplasmid was constructed by cloning CaMKIIα-EYFP into an AAV backboneusing MluI and EcoRI restriction sites. The maps are available online atwww.optogenetics.org, which are incorporated herein by reference.

Stereotactic injection and optical fiber placement: All surgeries wereperformed under aseptic conditions under stereotaxic guidance. Mice wereanaesthetized using 1.5-3.0% isoflourane. All coordinates are relativeto bregma in mm³. In all experiments, both in vivo and in vitro, viruswas delivered to the BLA only, and any viral expression in the CeArendered exclusion from all experiments. Cannula guides were beveled toform a 45-55 degree angle for the restriction of the illumination to theCeA. The short side of the beveled cannula guide was placedantero-medially, the long side of the beveled cannula shielded theposterior-lateral portion of the light cone, facing the oppositedirection of the viral injection needle. To preferentially targetBLA-CeL synapses, we restricted opsin gene expression to BLAglutamatergic projection neurons and restricted light delivery to theCeA. Control of BLA glutamatergic projection neurons was achieved usingan adeno-associated virus (AAV5) vector carrying light-activatedoptogenetic control genes under the control of a CaMKIIα promoter.Within the BLA, CaMKIIα is only expressed in glutamatergic pyramidalneurons, not in local interneurons⁴. Mice in the ChR2 Terminals and EYFPTerminals groups received unilateral implantations of beveled cannulaefor the optical fiber (counter-balanced for hemisphere), while mice inthe eNpHR 3.0 or respective EYFP group received bilateral implantationsof the beveled cannulae over the CeA (−1.06 mm anteroposterior (AP);±2.25 mm mediolateral (ML); and −4.4 mm dorsoventral (DV); PlasticsOne,Roanoke, Va.)³. Mice in the ChR2 Cell Bodies groups received unilateralimplantation of a Doric patchcord chronically implantable fiber(NA=0.22; Doric lenses, Quebec, Canada) over the BLA at (−1.6 mm AP;±3.1 mm ML; −4.5 mm DV)³. For all mice, 0.5 μl of purified AAV₅ wasinjected unilaterally or bilaterally in the BLA (±3.1 mm AP, 1.6 mm ML,−4.9 mm DV)³ using beveled 33 or 35 gauge metal needle facingposterolateral side to restrict the viral infusion to the BLA. 10 μlHamilton microsyringe (nanofil; WPI, Sarasota, Fla.) were used todeliver concentrated AAV solution using a microsyringe pump (UMP3; WPI,Sarasota, Fla.) and its controller (Micro4; WPI, Sarasota, Fla.). Then,0.5 μl of virus solution was injected at each site at a rate of 0.1 μlper min. After injection completion, the needle was lifted 0.1 mm andstayed for 10 additional minutes and then slowly withdrawn. One layer ofadhesive cement (C&B metabond; Parkell, Edgewood, N.Y.) followed bycranioplastic cement (Dental cement; Stoelting, Wood Dale, Ill.) wasused to secure the fiber guide system to the skull. After 20 min, theincision was closed using tissue adhesive (Vetbond; Fisher, Pittsburgh,Pa.). The animal was kept on a heating pad until it recovered fromanesthesia. A dummy cap (rat: C312G, mouse: C313G) was inserted to keepthe cannula guide patent. Behavioral and electrophysiologicalexperiments were conducted 4-6 weeks later to allow for viralexpression.

In vivo recordings: Simultaneous optical stimulation of central amygdala(CeA) and electrical recording of basolateral amygdala (BLA) of adultmale mice previously (4-6 weeks prior) transduced in BLA withAAV-CaMKIIα-ChR2-eYFP viral construct was carried out as describedpreviously (Gradinaru et al., J Neurosci, 27:14231-14238, 2007). Animalswere deeply anesthetized with isoflurane prior to craniotomy and hadnegative toe pinch. After aligning mouse stereotaxically and surgicallyremoving approximately 3 mm² skull dorsal to amygdala. Coordinates wereadjusted to allow for developmental growth of the skull and brain, asmice received surgery when they were 4-6 weeks old and experiments wereperformed when the mice were 8-10 weeks old (centered at −1.5 mm AP,±2.75 mm ML)³, a 1 Mohm 0.005-in extracellular tungsten electrode (A-Msystems) was stereotactically inserted into the craniotomized brainregion above the BLA (in mm: −1.65 AP, ±3.35 ML, −4.9 DV)³. Separately,a 0.2 N.A. 200 μm core diameter fiber optic cable (Thor Labs) wasstereotactically inserted into the brain dorsal to CeA (−1.1 AP, ±2.25ML, −4.2 DV)³. After acquiring a light evoked response, voltage rampswere used to vary light intensity during stimulation epochs (20 Hz, 5 mspulse width) 2 s in length. After acquiring optically evoked signal, theexact position of the fiber was recorded, the fiber removed from thebrain, inserted into a custom beveled cannula, reinserted to the sameposition, and the same protocol was repeated. In most trials, thefiber/cannula was then extracted from the brain, the cannula removed,and the bare fiber reinserted to ensure the fidelity of the populationof neurons emitting the evoked signal. Recorded signals were bandpassfiltered between 300 Hz and 20 kHz, AC amplified either 1000× or 10000×(A-M Systems 1800), and digitized (Molecular Devices Digidata 1322A)before being recorded using Clampex software (Molecular Devices).Clampex software was used for both recording field signals andcontrolling a 473 nm (OEM Laser Systems) solidstate laser diode sourcecoupled to the optrode. Light power was titrated between <1 mW (˜14mW/mm²) and 28 mW (˜396 mW/mm²) from the fiber tip and measured using astandard light power meter (ThorLabs). Electrophysiological recordingswere initiated approximately 1 mm dorsal to BLA after loweringisoflurane anesthesia to a constant level of 1%. Optrode was loweredventrally in ˜0.1 mm steps until localization of optically evokedsignal.

Behavioral assays: All animals used for behavior received viraltransduction of BLA neurons and the implantation enabling unilateral(for ChR2 groups and controls) or bilateral (for eNpHR3.0 groups andcontrols) light delivery. For behavior, multimode optical fibers (NA0.37; 300 μm core, BFL37-300; ThorLabs, Newton, N.J.) were precisely cutto the optimal length for restricting the light to the CeA, which wasshorter than the long edge of the beveled cannula, but longer than theshortest edge of the beveled cannula. For optical stimulation, the fiberwas connected to a 473 nm or 594 nm laser diode (OEM Laser Systems, EastLansing, Mich.) through an FC/PC adapter. Laser output was controlledusing a Master-8 pulse stimulator (A.M.P.I., Jerusalem, Israel) todeliver light trains at 20 Hz, 5 ms pulse-width for 473 nm light, andconstant light for 594 nm light experiments. All included animals hadthe center of the viral injection located in the BLA, though there wassometimes leak to neighboring regions or along the needle tract. Anycase in which there was any detectable viral expression in the CeA, theanimals were excluded. All statistically significant effects of lightwere discussed, and undiscussed comparisons did not show detectabledifferences.

The elevated plus maze was made of plastic and consisted of two lightgray open arms (30×5 cm), two black enclosed arms (30×5×30 cm) extendingfrom a central platform (5×5×5 cm) at 90 degrees in the form of a plus.The maze was placed 30 cm above the floor. Mice were individually placedin the center. 1-5 minutes were allowed for recovery from handlingbefore the session was initiated. Video tracking software (BiObserve,Fort Lee, N.J.) was used to track mouse location, velocity and movementof head, body and tail. All measurements displayed were relative to themouse body. Light stimulation protocols are specified by group.ChR2:BLA-CeA mice and corresponding controls groups (EYFP:BLA-CeA andChR2:BLA Somata) were singly-housed in a high-stress environment for atleast 1 week prior to anxiety assays: unilateral illumination of BLAterminals in the CeA at 7-8 mW (˜106 mW/mm² at the tip of the fiber,˜6.3 mW/mm² at CeL and ˜2.4 mW/mm² at the CeM) of 473 nm light pulsetrains (5 ms pulses at 20 Hz). For the ChR2 Cell Bodies group BLAneurons were directly illuminated with a lower light power becauseillumination with 7-8 mW induced seizure activity, so we unilaterallyilluminated BLA neurons at 3-5 mW (˜57 mW/mm²) of 473 nm light pulsetrains (5 ms pulses at 20 Hz). For the eNpHR 3.0 and corresponding EYFPgroup, all mice were group-housed and received bilateral viralinjections and bilateral illumination of BLA terminals in the CeA at 4-6mW (˜71 mW/mm² at the tip of the fiber, ˜4.7 mW/mm² at the CeL and ˜1.9mW/mm² at the CeM) of 594 nm light with constant illumination throughoutthe 5-min light on epoch. The 15-min session was divided into 35-minepochs, the first epoch there was no light stimulation (off), the secondepoch light was delivered as specified above (on), and the third epochthere was no light stimulation (off).

The open-field chamber (50×50 cm) and the open field was divided into acentral field (center, 23×23 cm) and an outer field (periphery).Individual mice were placed in the periphery of the field and the pathsof the animals were recorded by a video camera. The total distancetraveled was analyzed by using the same video-tracking software, Viewer²(BiObserve, Fort Lee, N.J.). The open field assessment was madeimmediately after the elevated-plus maze test. The open field testconsisted of an 18-min session in which there were six 3-min epochs. Theepochs alternated between no light and light stimulation periods,beginning with a light off epoch. For all analyses and charts where only“off” and “on” conditions are displayed, the 3 “off” epochs were pooledand the 3 “on” epochs were pooled.

For the glutamate receptor antagonist manipulation, a glutamateantagonist solution consisting of 22.0 mM of NBQX and 38.0 mM of D-APV(Tocris, Ellisville, Mo.) dissolved in saline (0.9% NaCl). 5-15 minbefore the anxiety assays, 0.3 μl of the glutamate antagonist solutionwas infused into the CeA via an internal infusion needle, inserted intothe same guide cannulae used for light delivery via optical fiber, thatwas connected to a 10-μl Hamilton syringe (nanofil; WPI, Sarasota,Fla.). The flow rate (0.1 μl per min) was regulated by a syringe pump(Harvard Apparatus, Mass.). Placements of the viral injection, guidecannula and chronically-implanted fiber were histologically verified asindicated in FIGS. 7 and 10.

Two-photon optogenetic circuit mapping and ex vivo electrophysiologicalrecording: Mice were injected with AAV5-CaMKIIα-ChR2-EYFP at 4 weeks ofage, and were sacrificed for acute slice preparation 4-6 weeks to allowfor viral expression. Coronal slices containing the BLA and CeA wereprepared to examine the functional connectivity between the BLA and theCeA. Two-photon images and electrophysiological recordings were madeunder the constant perfusion of aCSF, which contained (in mM): 126 NaCl,26 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, and 10 glucose. Allrecordings were at 32° C. Patch electrodes (4-6 MOhms) were filled (inmM): 10 HEPES, 4 Mg-ATP, 0.5 MgCl₂, 0.4 Na₃-GTP, 10 NaCl, 140 potassiumgluconate, and 80 Alexa-Fluor 594 hydrazide (Molecular Probes, EugeneOreg.). Whole-cell patch-clamp recordings were performed in BLA, CeL andCeM neurons, and cells were allowed to fill for approximately 30 minutesbefore imaging on a modified two-photon microscope (Prairie Microscopes,Madison Wis.) where two-photon imaging, whole-cell recording andoptogenetic stimulation could be done simultaneously. Series resistanceof the pipettes was usually 10-20 MOhms Blue light pulses were elicitedusing a 473 nm LED at −7 mW/mm² (Thorlabs, Newton N.J.) unless otherwisenoted. A Coherent Ti-Saphire laser was used to image both ChR2-YFP (940nm) and Alexa-Fluor 594 (800 nm). A FF560 dichroic with filters 630/69and 542/27 (Semrock, Rochester N.Y.) was also used to separate bothmolecules' emission. All images were taken using a 40×/0.8 NALUMPlanFL/IR Objective (Olympus, Center Valley Pa.). In order to isolatefibers projecting to CeL from the BLA and examine responses in the CeM,slices were prepared as described above with the BLA excluded fromillumination. Whole-cell recordings were performed in the CeM withillumination from the objective aimed over the CeL. To further ensureactivation of terminals from the BLA to CeL was selective, illuminationwas restricted to a ˜125 μm diameter around the center of the CeL. Here,blue light pulses were elicited using an XCite halogen light source(EXPO, Mississauga, Ontario) with a 470/3 filter at 6.5 mW/mm² coupledto a shutter (Uniblitz, Rochester N.Y.). For functional mapping, wefirst recorded from a BLA neuron expressing ChR2 and simultaneouslycollected electrophysiological recordings and filled the cell withAlexa-Fluor 594 hydrazide dye to allow for two-photon imaging.Two-photon z-stacks were collected at multiple locations along the axonof the filled BLA neuron. We then followed the axon of the BLA neuronprojecting to the CeL nucleus and recorded from a CeL neuron in the BLAterminal field. We then simultaneously recorded from a CeL neuron,filled the cell with dye and performed two-photon live imaging beforefollowing the CeL neuronal axons to the CeM. We then repeated thisprocedure in a CeM neuron, but moved the light back to the terminalfield in the CeL to mimic the preferential illumination of BLA-CeLsynapses with the same stimulation parameters as performed in vivo.Voltage-clamp recordings were made at both −70 mV, to isolate EPSCs, andat 0 mV, to isolate IPSCs. EPSCs were confirmed to be EPSCs via bathapplication of the glutamate receptor antagonists (n=5), NBQX (22 μm)and AP5 (38 μM), IPSCs were confirmed to be IPSCs via bath applicationof bicuculline (10 μM; n=2), which abolished them, respectively. We alsoperformed current-clamp recordings when the cell was resting atapproximately −70 mV.

For the characterization of optogenetically-driven antidromicstimulation in BLA axon terminals, animals were injected withAAV5-CaMKIIα-ChR2-EYFP at 4 weeks of age, and were sacrificed for acuteslice preparation 4-6 weeks to allow for viral expression. Slicepreparation was the same as above. To the aCSF we added 0.1 mMpicrotoxin, 10 μM CNQX and 25 μM AP5 (Sigma, St. Louis, Mo.). Whole-cellpatch-clamp recordings were performed in BLA neurons and were allowed tofill for approximately 30 minutes before two-photon imaging. Seriesresistance of the pipettes was usually 10-20 MOhms. All images weretaken using a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus, Center ValleyPa.). Blue light pulses were elicited using an XCite halogen lightsource (EXPO, Mississauga, Ontario) with a 470/30 filter at 6.5 mW/mm²coupled to a shutter (Uniblitz, Rochester N.Y.). Two-photon z-stackswere collected at multiple locations along the axon of the filled BLAneuron. Only neurons whose axons could be visualized for over ˜300 μmdiameter towards the CeL nucleus were included for the experiment, andneurons that had processes going in all directions were also excluded.Stimulation on/off axon was accomplished by moving the slice relative toa ˜125 μm diameter blue light spot. In order to calibrate the slice forcorrect expression, whole-cell patch-clamp was performed on a CeL celland a ˜125 μm diameter spot blue pulse was used to ensure that synapticrelease from the BLA terminals on to the CeL neuron was reliable.

For the dissection of direct and indirect projections to CeM, animalswere injected with AAV₅-CaMKIIα-ChR2-EYFP at 4 weeks of age, and weresacrificed for acute slice preparation 4-6 weeks to allow for viralexpression. Slice preparation was the same as above. Light was deliveredthrough a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus, Center ValleyPa.). Prior to whole cell patch clamping in the CeM nucleus, thelocation of the CeL nucleus was noted in order to revisit it with thelight spot restricted to this region. Whole-cell patch-clamp recordingswere performed in CeM neurons. Series resistance of the pipettes wasusually 10-20 MOhms. Blue light pulses were elicited using a XCitehalogen light source (EXPO, Mississauga, Ontario) with a 470/30 filterat 6.5 mW/mm² coupled to a shutter (Uniblitz, Rochester N.Y.). DuringCeM recordings, broad illumination (˜425-450 μm in diameter) of BLAterminals in the CeA and 20 Hz, 5 ms light train for 2 s was applied.Voltage-clamp recordings were made at 70 mV and 0 mV to isolate EPSCsand IPSCs respectively. Current-clamp recordings were also made. Then,illumination was moved to the CeL using a restricted light spot ˜125 μmin diameter. We again performed voltage clamp recordings at −70 mV and 0mV and used 20 Hz, 5 ms light train for 2 s. For the CeM neuron spikinginhibition experiments, in current-clamp, we applied the minimal currentstep required to induce spiking (˜60 pA) and simultaneously appliedpreferential illumination of ChR2-expressing BLA terminals in the CeLwith a 20 Hz, 5 ms light train for 2 s (mean over 6 sweeps per cell).For the experiments comparing the broad illumination of the BLA terminalfield centered in the CeM to selective illumination of BLA-CeLterminals, these conditions were performed in repeated alternation inthe same CeM cells (n=7).

To verify that terminal inhibition did not alter somatic spiking,animals were injected with AAV5-CaMKIIα-eNpHR3.0-EYFP at 4 weeks of age,and were sacrificed for acute slice preparation 4-6 weeks to allow forviral expression. Slice preparation was the same as above. Whole-cellpatch-clamp recordings were performed in BLA neurons and were allowed tofill for approximately 30 minutes. Light was delivered through a 40×/0.8NA LUMPlanFL/IR Objective (Olympus, Center Valley Pa.). Whole-cellpatch-clamp recordings were performed on BLA neurons. Series resistanceof the pipettes was usually 10-20 MOhms. Yellow light pulses wereelicited using a XCite halogen light source (EXPO, Mississauga, Ontario)with a 589/24 filter at 6.5 mW/mm² coupled to a shutter (Uniblitz,Rochester N.Y.). After patching, an unrestricted light spot (˜425-450microns in diameter) was placed over the BLA soma and a 1 s pulse wasapplied. Cells were excluded if the current recorded was under 600 pA ofhyperpolarizing current and the axon did not travel over ˜300 μm towardsthe CeL nucleus. The light spot was then restricted to −125 in diameter.On and off axon voltage clamp recordings were taken with a 1 s pulse oflight. For the current clamp recordings, action potentials weregenerated by applying 250 pA of current to the cell soma through thepatch pipette.

To demonstrate that selective illumination of eNpHR3.0-expressing BLAterminals reduced the probability of spontaneous vesicle release,animals were injected with AAV5-CaMKIIα-eNpHR3.0-EYFP at 4 weeks of age,and were sacrificed for acute slice preparation 4-6 weeks to allow forviral expression. Slice preparation was the same as above. Whole-cellpatch-clamp recordings were performed in central lateral neurons. Lightwas delivered through a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus,Center Valley Pa.). Series resistance of the pipettes was usually 10-20MOhms Yellow light pulses were elicited using a XCite halogen lightsource (EXPO, Mississauga, Ontario) with a 589/24 filter at 6.5 mW/mm²coupled to a shutter (Uniblitz, Rochester N.Y.). The light spot wasrestricted to ˜125 μm in diameter. Carbachol was added to the bath at aconcentration of 20 μM. After sEPSC activity increased in the CeLneuron, light pulses were applied ranging in times from 5 s to 30 s.

To demonstrate that selective illumination of eNpHR3.0-expressing BLAterminals could reduce the probability of vesicle release evoked byelectrical stimulation, animals were injected withAAV5-CaMKIIα-eNpHR3.0-EYFP at 4 weeks of age, and were sacrificed foracute slice preparation 4-6 weeks to allow for viral expression. Slicepreparation was the same as above. A bipolar concentric stimulationprobe (FHC, Bowdoin Me.) was placed in the BLA. Whole-cell patch-clamprecordings were performed in CeL neurons. Light was delivered through a40×/0.8 NA LUMPlanFL/IR Objective (Olympus, Center Valley Pa.). Seriesresistance of the pipettes was usually 10-20 MOhms. Amber light pulsesover the central lateral cell were elicited using a XCite halogen lightsource (EXPO, Mississauga, Ontario) with a 589/24 filter at 6.5 mW/mm²coupled to a shutter (Uniblitz, Rochester N.Y.). The light spot wasrestricted to ˜125 μm in diameter. Electrical pulses were delivered for40 seconds and light was delivered starting at 10 seconds and shut offat 30 seconds in the middle.

For the anatomical tracing experiments, neurons were excluded when thetraced axons were observed to be severed and all BLA neurons included inthe anatomical assay (FIG. 5 a-i) showed spiking patterns typical of BLApyramidal neurons¹⁸ upon a current step.

Slice immunohistochemistry: Anesthetized mice were transcardiallyperfused with ice-cold 4% paraformaldehyde (PFA) in PBS (pH 7.4) 100-110min after termination of in vivo light stimulation. Brains were fixedovernight in 4% PFA and then equilibrated in 30% sucrose in PBS. 40μm-thick coronal sections were cut on a freezing microtome and stored incryoprotectant at 4° C. until processed for immunohistochemistry.Free-floating sections were washed in PBS and then incubated for 30 minin 0.3% T×100 and 3% normal donkey serum (NDS). Primary antibodyincubations were performed overnight at 4° C. in 3% NDS/PBS (rabbitanti-c-fos 1:500, Calbiochem, La Jolla, Calif.; mouse anti-CaMKII 1:500,Abcam, Cambridge, Mass.). Sections were then washed and incubated withsecondary antibodies (1:1000) conjugated to Cy3 or Cy5 (JacksonLaboratories, West Grove, Pa.) for 3 hrs at room temperature. Followinga 20 min incubation with DAPI (1:50,000) sections were washed andmounted on microscope slides with PVD-DABCO.

Confocal microscopy and analysis: Confocal fluorescence images wereacquired on a Leica TCS SP5 scanning laser microscope using a 20×/0.70NAor a 40×/1.25NA oil immersion objective. Serial stack images covering adepth of 10 μm through multiple sections were acquired using equivalentsettings. The Volocity image analysis software (Improvision/PerkinElmer,Waltham, Mass.) calculated the number of c-fos positive cells per fieldby thresholding c-fos immunoreactivity above background levels and usingthe DAPI staining to delineate nuclei. All imaging and analysis wasperformed blind to the experimental conditions.

Statistics: For behavioral experiments and the ex vivo electrophysiologydata, binary comparisons were tested using nonparametric bootstrappedt-tests (paired or unpaired where appropriate)⁵, while hypothesesinvolving more than two group means were tested using linear contrasts(using the “boot” and “lme4” packages in R⁶, respectively); the latterwere formulated as contrasts between coefficients of a linearmixed-effects model (a “two-way repeated-measures ANOVA”) with the fixedeffects being the genetic or pharmacological manipulation and the lighttreatment (on or off). All hypothesis tests were specified a priori.Subjects were modeled as a random effects. For c-fos quantificationcomparisons, we used a one-way ANOVA followed by Tukey's multiplecomparisons test.

Plots of the data clearly show a relationship between observation meanand observation variance (that is, they are heteroskedastic; see forexample, FIG. 3 e and FIG. 5 j). We found that a standard square-roottransformation corrected this well. Additionally, eNpHR3.0 elevated plusmaze (EPM) data required detrending by a linear fit over time to accountfor a decrease in exploration behavior over time. As is standard for atwo-way linear mixed effects model (also known as a two-wayrepeated-measures ANOVA), we model (the square-root corrected value of)the kth observation in the ijth cell (y_(ijk)) as

√{square root over (y _(ijk))}=μ+c _(i) +t _(j)+(c:t)_(ij) +b _(j) +e_(ijk)  (1)

where

μ is the grand mean across all cells (where the ijth “cell” in thecollection of observations corresponding to the ith condition and jthtreatment)c_(i) is a fixed effect due to the ith animal condition acrosstreatments (for example, a genetic manipulation)t_(j) is a fixed effect due to the jth treatment across conditions (forexample, light on or light off)(c:t)_(ij) is a fixed effect due to the interaction of the ith conditionand jth treatment in the ijth cellb_(j) is a random effect corresponding to animals being used acrosstreatments, ande_(ijk) is an independent and identically distributed (i.i.d.) randomnormal disturbance in the ijkth observation with mean 0 and variance σ²,and independent of b_(j) for all j

Collecting the fixed effects into a 2-way analysis of variance (ANOVA)design matrix X

R^(nxp), dummy coding the random effects in a sparse matrix Z

R^(nxq), and letting {tilde over (y)}=√{square root over (y)} we canexpress the model in matrix form as

{tilde over (y)}=Xβ+Zb+e  (2)

where {tilde over (y)}ε

^(n), bε

^(q), and eε

^(n) are observations of random variables {tilde over (y)}, B, and εrespectively and our model assumes

-   -   B˜N(0, σ²Σ)    -   ε˜N(0, σ² I),ε⊥B    -   ({tilde over (y)}|B=b)˜N(Xβ+Zb, σ ² I)

where N (μ, Σ) denotes the multivariate Gaussian distribution with meanvector μ and variance-covariance matrix Σ, and ⊥ indicates that twovariables are independent. To estimate the coefficient vectors β

R^(p), b

R^(q), and the variance parameter σ and sparse (block-diagonal) relativevariance-covariance matrix Σ

R^(qxq), we use the lme4 package in R written by Douglas Bates andMartin Maechler, which first finds a linear change of coordinates that“spheres” the random effects and then finds the maximum likelihoodestimates for β, σ, and Σ using penalized iteratively reweightedleast-squares, exploiting the sparsity of the random effects matrix tospeed computation. For more details see the documentation accompanyingthe package in the lme4 repository at http://www.r-project.org/.

To solve for the maximum likelihood estimates, the design matrix X inequation 2 must be of full column rank. It is well known that this isnot the case for a full factorial design matrix with an intercept (as inequation 1), and thus linear combinations (“contrasts”) must be used todefine the columns of X in order for the fixed-effect coefficients to beestimable. As our designs are balanced (or nearly balanced), we usedorthogonal (or nearly orthogonal) Helmert contrasts between thecoefficients associated with light on as compared to light offconditions, terminal stimulation as compared to control conditions, andso on, as reported in the main text. Such contrasts allowed us tocompare pooled data (e.g., from several sequential light on vs. lightoff conditions) against each other within a repeated-measuresdesign—yielding improved parameter estimation and test power whileaccounting for within-animal correlations.

Results

BLA cells have promiscuous projections throughout the brain, includingto the bed nucleus of the stria terminalis (BNST), nucleus accumbens,hippocampus and cortex^(38, 43). To test whether BLA-CeL synapses couldbe causally involved in anxiety, it was therefore necessary to develop amethod to selectively control BLA terminals in the CeL, without directlyaffecting other BLA projections. To preferentially target BLA-CeLsynapses, we restricted opsin gene expression to BLA glutamatergicprojection neurons and restricted light delivery to the CeA. Control ofBLA glutamatergic projection neurons was achieved with anadeno-associated virus (AAV5) vector carrying light-activatedoptogenetic control genes under the control of a CaMKIIα promoter;within the BLA, CaMKIIα is only expressed in glutamatergic pyramidalneurons, not in local interneurons or intercalated cells⁴⁸. Topreferentially deliver light to the CeA projection, virus was deliveredunilaterally into the BLA under stereotaxic guidance (FIGS. 7 and 8)along with implantation of a beveled guide cannula over the CeL toprevent light delivery to the BLA and allow selective illumination ofthe CeA. Geometric and functional properties of the resulting lightdistribution were quantified both in vitro and in vivo, with in vivoelectrophysiological recordings to determine light power parameters forselective control of BLA terminals but not BLA cell bodies (FIG. 9).

To test the hypothesis that the BLA-CeA pathway could implement anendogenous mechanism for anxiolysis, we probed freely-moving mice underprojection-specific optogenetic control in two distinct andwell-validated anxiety assays: the elevated plus maze and the open fieldtest (FIG. 3 a-f). Mice display anxiety-related behaviors when exposedto open or exposed spaces, therefore increased time spent in the exposedarms of the elevated plus maze or in the center of the open fieldchamber indicates reduced anxiety^(49, 50). To test for both inductionand reversal of relevant behaviors, we first exposed mice to theelevated plus maze for three 5-min epochs, in which light was deliveredduring the second epoch only.

To determine whether the anxiolytic effect we observed would be specificto activation of BLA terminals in the CeA, and not BLA cells in general,we compared mice receiving projection-specific control (in theChR2:BLA-CeA group; FIG. 3 a) to both a negative control group receivingtransduction with a control virus given the same pattern of illumination(EYFP:BLA-CeA) and a positive control group transduced with theAAV-CaMKIIα-ChR2-EYFP virus in the BLA with a fiber implanted directlyover the BLA (ChR2:BLA Somata). For this group (ChR2:BLA Somata), lightstimulation did not elicit the anxiolysis observed in the ChR2:BLA-CeAgroup (FIGS. 3 b and c); indeed, the ChR2:BLA-CeA group spentsignificantly more time in open arms (t(42)=8.312; p<0.00001; FIG. 3b,c) during light-induced activation of BLA terminals in the CeA, incomparison to controls (EYFP:BLA-CeA and ChR2:BLA Somata groups). TheChR2:BLA-CeA mice also showed an increase in the probability of enteringan open arm rather than a closed arm, from the choice point of thecenter of the maze (FIG. 3 c inset), indicating an increased probabilityof selecting the normally anxiogenic environment.

We also probed mice on the open field arena for six 3-minute epochs,again testing for reversibility by alternating between no light (off)and light stimulation (on) conditions. Experimental (ChR2:BLA-CeA) micedisplayed an immediate, robust, and reversible light-induced anxiolyticresponse as measured by the time in center of the open field chamber(FIGS. 3 d and e), while mice in the EYFP:BLA-CeA and ChR2:BLA Somatagroups did not (FIG. 3 e). Light stimulation did not significantly alterlocomotor activity (FIG. 3 f). While there was no detectable differenceamong groups in the off conditions, there was a significant increase incenter time of the open field spent by mice in the ChR2:BLA-CeA grouprelative to the EYFP:BLA-CeA or ChR2:BLA Somata groups during the onconditions (t(105)=4.96178; p<0.0001 for each contrast). We concludedthat selective stimulation of BLA projections to the CeA, but not BLAsomata, produces an acute, rapidly reversible anxiolytic effect,supporting the hypothesis that the BLA-CeL-CeM pathway could represent anative microcircuit for anxiety control.

We next investigated the physiological basis of this light-inducedanxiolytic effect. Glutamatergic neurons in the BLA send robustexcitatory projections to CeL neurons as well as to CeM neurons³⁸;however, not only are the CeM synapses distant from the light source(FIG. 8), but also any residual direct excitation of these CeM neuronswould be expected to result in an anxiogenic, rather than an anxiolytic,effect¹². However, CeL neurons exert strong inhibition onto thesebrainstem-projecting CeM output neurons^(32, 35, 40), and we thereforehypothesized that illumination of BLA terminals in the CeA couldactivate BLA-CeL neurons and thereby elicit feed-forward inhibition ontoCeM neurons and implement the observed anxiolytic phenomenon.

To confirm the operation of this optogenetically-defined projection, weundertook in vivo experiments, with light delivery protocols matched tothose delivered in the behavioral experiments, and activity-dependentimmediate early gene (c-fos) expression analysis as the readout toverify the pattern of neuronal activation (FIG. 3 g-k). Under blindedconditions, we quantified the proportion of neurons in the BLA, CeL andCeM (FIG. 3 i-k) for ChR2:BLA-CeA, EYFP:BLA-CeA and ChR2:BLA Somatagroups that expressed EYFP or showed c-fos immunoreactivity. Virusexpression under the CaMKIIα promoter in the BLA targeted glutamatergicneurons⁴⁷, and we did not observe EYFP expression in local interneuronsnor intercalated cells (FIG. 10). No significant differences amonggroups were detected in the proportion of EYFP-positive cells withineach region (FIG. 3 g-k), but we found a significantly higher proportionof c-fos positive BLA cells in the ChR2:BLA Somata group, relative toChR2:BLA-CeA or EYFP:BLA-CeA groups (FIG. 3 i; p<0.01 and p<0.05,respectively). There was no detectable difference in c-fos between theChR2:BLA-CeA and EYFP:BLA-CeA groups, indicating that the beveledcannula shielding effectively prevented direct illumination to BLA cellbodies. A significantly higher proportion of CeL neurons expressed c-fosin the ChR2:BLA-CeA group relative to the EYFP:BLA-CeA group (p<0.05),but not the ChR2:BLA Somata group (FIG. 3 j). Thus, selectiveillumination of BLA terminals expressing ChR2 in the CeA led topreferential activation of CeL neurons, without activating BLA somata.In the CeM, we found twice as many c-fos positive neurons (relative tototal neurons) in the ChR2:BLA Somata group than in the ChR2:BLA-CeA(FIG. 3 k), consistent with anatomical projections, as LA neuronsselectively innervate CeL neurons, while neurons in the BL and BM nucleiof the amygdala have monosynaptic projections to both the CeL and theCeM^(38, 43, 51). Together, these data reveal that the in vivoillumination that triggers an acute anxiolytic behavioral phenotypeimplements selective illumination of BLA-CeL synapses without activatingBLA cell bodies.

To test the hypothesis that selective illumination of BLA terminals inthe CeL induces feed-forward inhibition of CeM output neurons, wecombined whole-cell patch-clamp recording with live two-photon imagingto visualize the microcircuit while simultaneously probing thefunctional relationships among these cells during projection-specificoptogenetic control (FIG. 4 a-f). While the light-stimulation parametersused in vivo were delivered via a fiber optic and the parameters used inour ex vivo experiments were delivered onto acute slices, we matched thelight power density at our target location ˜6 mW/mm². A two-photon imageof the BLA-CeL-CeM circuit is shown in FIG. 4 a, with all three cellsimaged from the same slice (FIG. 4 a). The BLA neuron expressingChR2-EYFP showed robust, high-fidelity spiking to direct illuminationwith 20 Hz, 5 ms pulses of 473 nm light (FIG. 4 b). A representativetrace from a CeL neuron, recorded during illumination of the terminalfield of BLA neurons expressing ChR2-EYFP, demonstrates the typicalexcitatory responses seen in CeL (FIG. 4 c), with population summariesrevealing that spiking fidelity was steady throughout the 40-pulse lighttrain and that responding cells include both weakly and strongly-excitedCeL cells (n=16; FIG. 4 c). To test whether illumination of BLA-CeLsynapses would be functionally significant at the level of blockingspiking in CeM cells due to the robust feed-forward inhibition from CeLneurons, we recorded from CeM neurons while selectively illuminatingBLA-CeL synapses (FIG. 4 d). Indeed, we observed potent spikinginhibition (F_(2,11)=15.35, p=0.0044) in the CeM due to lightstimulation of BLA terminals in the CeL (FIG. 4 d; spikes per secondbefore (49±9.0), during (1.5±0.87), and after (33±8.4) illumination;mean±s.e.m). Next, FIG. 4 e shows CeM responses recorded duringillumination of the terminal field of BLA neurons in the CeM expressingChR2-EYFP, and the combined excitatory and inhibitory input. Populationsummaries from voltage-clamp recordings indicated that latencies ofEPSCs were shorter than those of the disynaptic IPSCs, as expected, andthat the mean IPSC amplitude was greater than mean EPSC amplitude(recorded at 0 and −70 mV, respectively; FIG. 4 e). Importantly, thevery same CeM neurons (n=7) yielded net excitation with broadillumination of BLA inputs to the CeM (FIG. 4 e), but displayed netinhibition with selective illumination of BLA inputs to the CeL (FIG. 4f) in a repeatable fashion with alternation between sites. Thisdemonstrates that the balance of direct and indirect inputs from the BLAto the CeM can modulate CeM output. Together, these data reveal astructurally- and functionally-identified physiological microcircuit,whereby selective illumination of BLA terminals in the CeA activatesBLA-CeL synapses, thus increasing feed-forward inhibition from CeLneurons onto the brainstem-projecting CeM neurons.

To further elucidate the amygdalar microcircuits underlying thisanxiolytic effect, we carefully dissected the anatomical and functionalproperties governing this phenomenon. While some efforts to map theprojections of BLA collaterals in the CeA have been made in the rat, weempirically tested whether overlapping or distinct populations of BLAneurons projected to the CeL and CeM (FIG. 5 a,b). A noteworthy caveatis that we visualized these neurons in ˜350 um thick coronal sectionsand while every attempt was made to exclude neurons in which the axonswere severed, we cannot exclude the possibility that this occurred norcan we deny that this induced some sampling bias for BLA neurons closerto the CeA. FIG. 5 a summarizes the anatomical projections of the BLAneurons sampled (n=18) and shows that the 44% of neurons projected tothe CeL alone and 17% projected to the CeM alone. However, a minority ofBLA cells (n=1; 6%), projected to both the CeL and the CeM, one of whichsent separate collaterals to the CeL and CeM and one of which sent acollateral that sent branches to the CeL and CeM. FIG. 5 b shows the2-photon image of each cell sampled, all of which showed spikingpatterns typical of BLA pyramidal neurons upon a current step.

Next, as our c-fos assays suggested that illumination of BLA terminalsin the CeL were sufficient to excite CeL neurons, but not BLA neuronsthemselves, we sought to confirm this hypothesis with whole-cellrecordings. With electrical stimulation, depolarization of axonterminals leads to antidromic spiking at the cell soma. However, therehas been evidence that optogenetically-induced depolarization functionsvia a distinct mechanism. To evaluate the properties ofoptogenetically-induced terminal stimulation in this amygdalarmicrocircuit, we recorded from BLA pyramidal neurons expressing ChR2 andmoved a light spot (˜120 μm in diameter) in 100 μm steps from the cellsoma, both in a direction over a visually-identified axon collateral andin a direction where there was no axon (FIG. 5 c). The spike fidelity ofthe BLA neuron given a 20 Hz train of light at each distance from thesoma is summarized in FIG. 5 d, while the depolarizing current issummarized in FIG. 5 e. In all preparations, we confirmed that the lightstimulation parameters used were sufficient to elicit high-fidelityspiking at the BLA cell soma (FIG. 5 f) and reliable vesicle release atBLA terminals as shown by recordings from a postsynaptic CeL neuron(FIG. 5 g; FIG. 15). In contrast, when recording from the same BLAneurons with the light spot 300 um away from the cell soma we did notobserve reliable action potential induction, regardless of whether wewere over an axon (FIG. 5 h) or not (FIG. 5 i). This absence ofantidromic spiking was observed even upon bath application of GABA andAMPA receptor antagonists (n=7), thus excluding the possiblecontribution of local inhibitory constraints. While we demonstrate thatoptogenetically-induced vesicle release can occur in the absence ofantidromic stimulation in BLA pyramidal neurons, it is possible that atantidromic stimulation could be achieved with greater light powerdensity than we used here (˜6 mW/mm²) Thusfar, we have demonstrated thatthe populations of BLA neurons projecting to the CeL and the CeM arelargely distinct and that illumination of BLA-CeL synapses inducesvesicle release and CeL excitation without strong activation of BLAsomata themselves.

Finally, we further explored the mechanism with in vivo pharmacologicalanalysis in the setting of projection-specific optogenetic control. Todetermine whether the anxiolytic effect we observed could be due to theselective activation of BLA-CeL synapses alone, and not BLA fiberspassing through the CeA, nor back-propagation of action potentials toBLA cell bodies which then would innervate all BLA projection targetregions, we tested whether local glutamate receptor antagonism wouldattenuate light-induced anxiolytic effects. This question is ofsubstantial interest since lesions in the CeA that alter anxiety areconfounded by the likelihood of ablation of BLA projections to the BNSTwhich pass through CeA⁶. We unilaterally transduced BLA neurons withAAV-CaMKIIα-ChR2-EYFP and implanted beveled cannulae to implementselective illumination of BLA terminals in the CeA as before (n=8; FIG.8), and tested mice on the elevated plus maze and open field test. Inthis case, however, we infused either the glutamate antagonists NBQX andAP5 using the optical fiber guide cannula, or saline control ondifferent trials in the same animals, with trials counter-balanced fororder. Confirming a local synaptic mechanism rather than control offibers of passage, for the same mice and light stimulation parameters,local glutamate receptor antagonism in the CeA abolished light-inducedreductions in anxiety on both the elevated plus maze (FIG. 5 k) and theopen field test (FIG. 5 j). Importantly, in control experiments, drugtreatment did not impair locomotor activity (FIG. 11), and in acuteslices time-locked light-evoked excitatory responses were abolished uponbath application of NBQX and AP5 (FIG. 12). Together these data indicatethat the light-induced anxiolytic effects we observed were caused by theactivation of BLA-CeL synapses, and not attributable to BLA projectionsto distal targets passing through the CeA.

In a final series of experiments, to determine if endogenousanxiety-reducing processes could be blocked by selectively inhibitingthis pathway, we tested whether the selective inhibition of theseoptogenetically defined synapses could reversibly increase anxiety. Weperformed bilateral viral transduction of either eNpHR3.0, alight-activated chloride pump which hyperpolarizes neuronal membranesupon illumination with amber light²⁵, or EYFP alone, both under theCaMKIIα promoter in the BLA, and implanted bilateral beveled guidecannulae to allow selective illumination of BLA terminals in the CeA(FIG. 6 a; FIG. 13). eNpHR3.0 expression was restricted to glutamatergicCaMKIIα-positive neurons in the BLA (FIG. 6 b). The eNpHR3.0:BLA-CeAgroup only showed significantly elevated levels of c-fos expression,relative to the EYFP:BLA-CeA bil and eNpHR 3.0:Soma groups, in the CeM(p<0.05; FIG. 6 c-e), consistent with the hypothesis that selectiveinhibition of BLA terminals in the CeA suppresses feed-forwardinhibition from CeL neurons to CeM neurons, thus increasing CeMexcitability and the downstream processes leading to increased anxietyphenotypes. Importantly, inhibition of BLA somata did not induce ananxiogenic response, likely due to the simultaneous decrease in directBLA-CeM excitatory input. We also found that the eNpHR3.0:BLA-CeA groupshowed a significant reduction in open arm time and probability of openarm entry on the elevated plus maze during light-on epochs, but notlight-off epochs, relative to the EYFP and Soma groups (FIG. 6 f,g),without altering locomotor activity (FIG. 16). The eNpHR3.0:BLA-CeAgroup also showed a significant reduction in center time uponillumination with 594 nm light, relative to the EYFP and Soma groups(statistics, p=0.002; FIG. 6 h,i) Finally, we also demonstrate thatselective illumination of eNpHR3.0-expressing axon terminals can reducethe probability of both spontaneously occurring (FIG. 6 j-l) and evoked(FIG. 6 m-p) vesicle release, without preventing spiking at the cellsoma (FIG. 14). These data demonstrate that selective inhibition of BLAterminals in the CeA induces an acute increase in anxiety-likebehaviors.

CONCLUSIONS

In these experiments, we have identified the BLA-CeL pathway as anendogenous neural substrate for bidirectionally modulating theunconditioned expression of anxiety. While we identify the BLA-CeLpathway as the critical substrate rather than BLA fibers passing throughthe CeL, it is likely that other downstream circuits, such as CeAprojections to the BNST play an important role in the expression ofanxiety or anxiety-related behaviors^(4, 6, 13). Indeed, our findingsmay support the notion that corticotrophin releasing hormone (CRH)networks in the BNST can be critically involved in modulatinganxiety-related behaviors^(6, 52), as the CeL is a primary source of CRHfor the BNST⁵³.

Other neurotransmitters and neuromodulators may modulate or gate effectson distributed neural circuits, including serotonin^(54, 55),dopamine⁵⁶, acetylcholine⁵⁷, glycine⁵⁸, GABA¹³ and CRH⁵⁹. The neuralcircuitry converging to and diverging from this pathway will providemany opportunities for modulatory control, as parallel or downstreamcircuits of the BLA-CeL synapse likely contribute to modulate theexpression of anxiety phenotypes^(6, 56). Moreover, upstream of theamygdala, this microcircuit is well-positioned to be recruited bytop-down cortical control from regions important for processing fear andanxiety, including the prelimbic, infralimbic and insular cortices thatprovide robust innervation to the BLA and CeL.^(4, 13, 23, 60).

Our examination of the BLA anatomy suggests that the populations of BLAneurons projecting to CeL and CeM neurons are largely non-overlapping.In natural states, the CeL-projecting BLA neurons may exciteCeM-projecting BLA neurons in a microcircuit homeostatic mechanism. Thismay also represent a potential mechanism underlying anxiety disorders,when there are synaptic changes that skew the balance of the circuit toallow uninhibited CeM activation.

Together, the data presented here support identification of the BLA-CeLsynapse as a critical circuit element both necessary and sufficient forthe expression of endogenous anxiolysis in the mammalian brain,providing a novel source of insight into anxiety as well as a new kindof treatment target, and demonstrate the importance of resolvingspecific projections in the study of neural circuit function relevant topsychiatric disease.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

All references, publications, and patent applications disclosed hereinare hereby incorporated by reference in their entirety.

REFERENCES

-   1. Lieb, R. Anxiety disorders: clinical presentation and    epidemiology. Handb Exp Pharmacol, 405-432 (2005).-   2. Kessler, R. C., et al. Lifetime prevalence and age-of-onset    distributions of DSM-IV disorders in the National Comorbidity Survey    Replication. Arch Gen Psychiatry 62, 593-602 (2005).-   3. Koob, G. F. Brain stress systems in the amygdala and addiction.    Brain Res 1293, 61-75 (2009).-   4. Ressler, K. J. & Mayberg, H. S. Targeting abnormal neural    circuits in mood and anxiety disorders: from the laboratory to the    clinic. Nat Neurosci 10, 1116-1124 (2007).-   5. Vanderschuren, L. J. & Everitt, B. J. Behavioral and neural    mechanisms of compulsive drug seeking. Eur J Pharmacol 526, 77-88    (2005).-   6. Davis, M., Walker, D. L., Miles, L. & Grillon, C. Phasic vs    sustained fear in rats and humans: role of the extended amygdala in    fear vs anxiety. Neuropsychopharmacology 35, 105-135.-   7. Ehrlich, I., et al. Amygdala inhibitory circuits and the control    of fear memory. Neuron 62, 757-771 (2009).-   8. Han, J. H., et al. Selective erasure of a fear memory. Science    323, 1492-1496 (2009).-   9. Herry, C., et al. Switching on and off fear by distinct neuronal    circuits. Nature 454, 600-606 (2008).-   10. LeDoux, J. The emotional brain, fear, and the amygdala. Cell Mol    Neurobiol 23, 727-738 (2003).-   11. Maren, S. & Quirk, G. J. Neuronal signaling of fear memory. Nat    Rev Neurosci 5, 844-852 (2004).-   12. Pare, D., Quirk, G. J. & Ledoux, J. E. New vistas on amygdala    networks in conditioned fear. J Neurophysiol 92, 1-9 (2004).-   13. Shin, L. M. & Liberzon, I. The neurocircuitry of fear, stress,    and anxiety disorders. Neuropsychopharmacology 35, 169-191.-   14. Davis, M. The role of the amygdala in conditioned and    unconditioned fear and anxiety. in The Amygdala (ed. A. JP) p.    213-288 (Oxford University Press, Oxford, UK, 2000).-   15. Killcross, S., Robbins, T. W. & Everitt, B. J. Different types    of fear-conditioned behaviour mediated by separate nuclei within    amygdala. Nature 388, 377-380 (1997).-   16. Tye, K. M. & Janak, P. H. Amygdala neurons differentially encode    motivation and reinforcement. J Neurosci 27, 3937-3945 (2007).-   17. Tye, K. M., Stuber, G. D., de Ridder, B., Bonci, A. &    Janak, P. H. Rapid strengthening of thalamo-amygdala synapses    mediates cue-reward learning. Nature 453, 1253-1257 (2008).-   18. Bahi, A., Mineur, Y. S. & Picciotto, M. R. Blockade of protein    phosphatase 2B activity in the amygdala increases anxiety- and    depression-like behaviors in mice. Biol Psychiatry 66, 1139-1146    (2009).-   19. Davis, M. Are different parts of the extended amygdala involved    in fear versus anxiety? Biol Psychiatry 44, 1239-1247 (1998).-   20. Etkin, A., et al. Individual differences in trait anxiety    predict the response of the basolateral amygdala to unconsciously    processed fearful faces. Neuron 44, 1043-1055 (2004).-   21. Kalin, N. H., Shelton, S. E. & Davidson, R. J. The role of the    central nucleus of the amygdala in mediating fear and anxiety in the    primate. J Neurosci 24, 5506-5515 (2004).-   22. Roozendaal, B., McEwen, B. S. & Chattarji, S. Stress, memory and    the amygdala. Nat Rev Neurosci (2009).-   23. Stein, M. B., Simmons, A. N., Feinstein, J. S. & Paulus, M. P.    Increased amygdala and insula activation during emotion processing    in anxiety-prone subjects. Am J Psychiatry 164, 318-327 (2007).-   24. Boyden, E. S., Zhang, F., Bamberg, E, Nagel, G. & Deisseroth, K.    Millisecond-timescale, genetically targeted optical control of    neural activity. Nat Neurosci 8, 1263-1268 (2005).-   25. Gradinaru, V., at aL Molecular and cellular approaches for    diversifying and extending optogenetics. Cell 141, 154-165.-   26. Nagel, G., at al. Channelrhodopsin-2, a directly light-gated    cation-selective membrane channel Proc Natl Acad Sci USA 100,    13940-13945 (2003).-   27. Fraser, A. D. Use and abuse of the benzodiazepines. Ther Drug    Monit 20, 481-489 (1998).-   28. Woods, J. H., Katz, J. L. & Winger, G. Benzodiazepines: use,    abuse, and consequences. Pharmacol Rev 44, 151-347 (1992).-   29. Hovatta, I. & Barlow, C. Molecular genetics of anxiety in mice    and men. Anti Med 40, 92-109 (2008).-   30. Hovatta, I., et al. Glyoxalase 1 and glutathione reductase 1    regulate anxiety in mice. Nature 438, 662-666 (2005).-   31. Blanchard, R. J., Yudko, E. B., Rodgers, R. J. &    Blanchard, D. C. Defense system psychopharmacology: an ethological    approach to the pharmacology of fear and anxiety. Behav Brain Res    58, 155-165 (1993).-   32. LeDoux, J. E., Iwata, J., Cicchetti, P. & Reis, D. J. Different    projections of the central amygdaloid nucleus mediate autonomic and    behavioral correlates of conditioned fear. J Neurosci 8, 2517-2529    (1988).-   33. Carlson, J. lrnmunocytochemical localization of glutamate    decarboxylase in the rat basolateral amygdaloid nucleus, with    special reference to GABAergic innervation of amygdalostriatal    projection neurons. J Comp Neurol 273, 513-526 (1988).-   34. Smith, Y. & Pare, D. Intra-amygdaloid projections of the lateral    nucleus in the cat: PHA-L anterograde labeling combined with    postembedding GABA and glutamate immunocytochemistry. J Comp Neurol    342, 232-248 (1994).-   35. McDonald, A. J. Cytoarchitecture of the central amygdaloid    nucleus of the rat. J Comp Neurol 208, 401-418 (1982).-   36. Bissiere, S., Humeau, Y. & Luthi, A. Dopamine gates LTP    induction in lateral amygdala by suppressing feedforward inhibition.    Nat Neurosci 6, 587-592 (2003).-   37. Marowsky, A., Yanagawa, Y., Obata, K. & Vogt, K. E. A    specialized subclass of interneurons mediates dopaminergic    facilitation of amygdala function. Neuron 48, 1025-1037 (2005).-   38. Pitkanen, A. Connectivity of the rat amygdaloid complex. in The    Amygdala (ed. A. JP) p. 31-99 (Oxford University Press, Oxford, UK,    2000).-   39. Krettek, J. E. & Price, J. L. Amygdaloid projections to    subcortical structures within the basal forebrain and brainstem in    the rat and cat. J Comp Neurol 178, 225-254 (1978).-   40. Petrovich, G. D. & Swanson, L. W. Projections from the lateral    part of the central amygdalar nucleus to the postulated fear    conditioning circuit. Brain Res 763, 247-254 (1997).-   41. LeDoux, J. E., Cicchetti, P., Xagoraris, A. & Romanski, L. M.    The lateral amygdaloid nucleus: sensory interface of the amygdala in    fear conditioning. J Neurosci 10, 1062-1069 (1990).-   42. Krettek, J. E. & Price, J. L. A description of the amygdaloid    complex in the rat and cat with observations on intra-amygdaloid    axonal connections. J Comp Neurol 178, 255-280 (1978).-   43. Petrovich, G. D., Risold, P. Y. & Swanson, L. W. Organization of    projections from the basomedial nucleus of the amygdala: a PHAL    study in the rat. J Comp Neural 374, 387-420 (1996).-   44. Pare, D. & Smith, Y. The intercalated cell masses project to the    central and medial nuclei of the amygdala in cats. Neuroscience 57,    1077-1090 (1993).-   45. Likhtik, E., Popa, D., Apergis-Schoute, J., Fidacaro, G. A. &    Pare, D. Amygdala intercalated neurons are required for expression    of fear extinction. Nature 454, 642-645 (2008).-   46. Jolkkonen, E. & Pitkanen, A. Intrinsic connections of the rat    amygdaloid complex: projections originating in the central nucleus.    J Comp Neurol 395, 53-72 (1998).-   47. Amano, T., Unal, C. T. & Pare, D. Synaptic correlates of fear    extinction in the amygdala. Nat Neurosci 13, 489-494.-   48. McDonald, A. J., Muller, J. F. & Mascagni, F. GABAergic    innervation of alpha type II calcium/calmodulin-dependent protein    kinase immunoreactive pyramidal neurons in the rat basolateral    amygdala. J Comp Neurol 446, 199-218 (2002).-   49. Choleris, E., Thomas, A. W., Kavaliers, M. & Prato, F. S. A    detailed ethological analysis of the mouse open field test: effects    of diazepam, chlordiazepoxide and an extremely low frequency pulsed    magnetic field. Neurosci Biobehav Rev 25, 235-260 (2001).-   50. Pellow, S., Chopin, P., File, S. E. & Briley, M. Validation of    open:closed arm entries in an elevated plus-maze as a measure of    anxiety in the rat. J Neurosci Methods 14, 149-167 (1985).-   51. Sah, P. & Lopez De Armentia, M. Excitatory synaptic transmission    in the lateral and central amygdala. Ann N Y Acad Sci 985, 67-77    (2003).-   52. Davis, M. & Shi, C. The extended amygdala: are the central    nucleus of the amygdala and the bed nucleus of the stria terminalis    differentially involved in fear versus anxiety? Ann N Y Acad Sci    877, 281291 (1999).-   53. Sakanaka, M., Shibasaki, T. & Lederis, K. Distribution and    efferent projections of corticotropin-releasing factor-like    immunoreactivity in the rat amygdaloid complex. Brain Res 382,    213-238 (1986).-   54. Holmes, A., Yang, R. J., Lesch, K. P., Crawley, J. N. &    Murphy, D. L. Mice lacking the serotonin transporter exhibit    5-HT(1A) receptor-mediated abnormalities in tests for anxiety-like    behavior. Neuropsychopharmacology 28, 2077-2088 (2003).-   55. Lesch, K. P., et al. Association of anxiety-related traits with    a polymorphism in the serotonin transporter gene regulatory region.    Science 274, 1527-1531 (1996).-   56. Graybiel, A. M. & Rauch, S. L. Toward a neurobiology of    obsessive-compulsive disorder. Neuron 28, 343-347 (2000).-   57. Picciotto, M. R., Brunzell, D. H. & Caldarone, B. J. Effect of    nicotine and nicotinic receptors on anxiety and depression.    Neuroreport 13, 1097-1106 (2002).-   58. Snyder, S. H. & Enna, S. J. The role of central glycine    receptors in the pharmacologic actions of benzodiazepines. Adv    Biochem Psychopharmacol, 81-91 (1975).-   59. Lesscher, H. M., et al. Amygdala protein kinase C epsilon    regulates corticotropin-releasing factor and anxiety-like behavior.    Genes Brain Behav 7, 323-333 (2008).-   60. Milad, M. R., Rauch, S. L., Pitman, R. K. & Quirk, G. J. Fear    extinction in rats: implications for human brain imaging and anxiety    disorders. Biol Psycho! 73, 61-71 (2006).

1-18. (canceled)
 19. A method for alleviating anxiety in an individual,the method comprising: (a) administering to the individual an effectiveamount of a recombinant expression vector comprising a nucleic acidencoding a light-responsive opsin, wherein the nucleic acid is operablylinked to a promoter that controls the specific expression of the opsinin the glutamatergic pyramidal neurons of the basolateral amygdala(BLA), wherein the opsin is expressed in the glutamatergic pyramidalneurons of the BLA, wherein the opsin is an opsin that inducesdepolarization by light; and (b) selectively illuminating the opsin inthe glutamatergic pyramidal neurons in the central nucleus of theamygdala (CeA) to alleviate anxiety.
 20. The method of claim 19, whereinthe opsin is selected from the group consisting of ChR2, VChR1, andDChR.
 21. A method for inducing anxiety in a non-human animal, themethod comprising: (a) administering to the non-human animal aneffective amount of a recombinant vector comprising a nucleic acidencoding an opsin, wherein the nucleic acid is operably linked to apromoter that controls the specific expression of the opsin in theglutamatergic pyramidal neurons of the basolateral amygdala (BLA),wherein the opsin is expressed in the glutamatergic pyramidal neurons ofthe BLA, wherein the opsin is an opsin that induces hyperpolarization bylight; and (b) selectively illuminating the opsin in the glutamatergicpyramidal neurons in the central nucleus of the amygdala (CeA) to induceanxiety. 22-31. (canceled)
 32. The method of claim 19, comprisingselectively illuminating the centrolateral nuclei (CeL).
 33. The methodof claim 19, wherein the opsin comprises an amino acid sequence havingat least 90% amino acid sequence identity to any one of SEQ ID NOs:6-11.
 34. The method of claim 19, wherein the opsin comprises an aminoacid sequence having at least 90% amino acid sequence identity to SEQ IDNO:6.
 35. The method of claim 19, wherein the opsin comprises an aminoacid sequence having at least 90% amino acid sequence identity to SEQ IDNO:7.
 36. The method of claim 19, wherein the opsin comprises an aminoacid sequence having at least 90% amino acid sequence identity to SEQ IDNO:8.
 37. The method of claim 19, wherein the opsin comprises an aminoacid sequence having at least 90% amino acid sequence identity to SEQ IDNO:9.
 38. The method of claim 19, wherein the opsin comprises an aminoacid sequence having at least 90% amino acid sequence identity to SEQ IDNO:10.
 39. The method of claim 19, wherein the opsin comprises an aminoacid sequence having at least 90% amino acid sequence identity to SEQ IDNO:11.
 40. The method of claim 19, wherein the recombinant vector is anadeno-associated vector, a retroviral vector, an adenoviral vector, or alentiviral vector.
 41. The method of claim 19, wherein the promoter is aCaMKIIα promoter.
 42. The method of claim 19, wherein said illuminationis carried out with an optical fiber.
 43. The method of claim 21,wherein the opsin is selected from the group consisting of NpHR, BR, AR,and GtR3.
 44. The method of claim 21, wherein the opsin comprises anamino acid sequence having at least 90% amino acid sequence identity toone of SEQ ID NOs: 1-4.